Methods of treating cancer in biomarker-identified patients with non-covalent inhibitors of cyclin-dependent kinase 7 (cdk7)

ABSTRACT

The present invention relates to methods of identifying patients suffering from various types of cancer who are more likely to respond to treatment with a CDK7 inhibitor conforming to structural Formula (I), (Ia), a species thereof, or a specified form thereof (as described herein), either when administered or used alone or in combination with a second therapeutic agent (e.g., another anti-cancer therapy). Patients are identified based on one or more features (e.g., gene copy number or expression level) of certain biomarkers (e.g., RB1 or another member of the E2F pathway). In addition, the present invention relates to methods of treating an identified patient with a compound conforming to structural Formula (I), (Ia), a species thereof, or a specified form thereof, either alone or in combination with a second therapeutic agent. In another aspect, the present invention features kits including instructions for treating a patient identified as described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.provisional application No. 62/754,398, filed Nov. 1, 2018; U.S.provisional application No. 62/877,189, filed Jul. 22, 2019; U.S.provisional application No. 62/915,983, filed Oct. 16, 2019, and U.S.provisional application No. 62/927,469, filed Oct. 29, 2019. The contentof each of these prior applications is hereby incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

The long evolution of healthcare has reached a point in time where thepromise of biomarker analysis is beginning to be realized. Whenphysicians can stratify patients, even those who share many similarphysiological traits and exhibit common symptoms of a given disease,into more specific groups, they can better tailor treatment and optimizethe outcome for each patient. However, it is challenging to developmolecular diagnostics and few are commercially available.

SUMMARY OF THE INVENTION

The present invention features, inter alia, diagnostic methods foridentifying cancer patients for treatment with a non-covalent CDK7inhibitor described herein (i.e., diagnostic methods for selecting apatient for treatment) and methods for treating identified patients withsuch an inhibitor, either alone or in combination with one or moreadditional therapeutic agents (e.g., a second anti-cancer agent), asdescribed further below. The diagnostic methods include a step ofidentifying a patient suffering from a cancer that is likely to respondwell to treatment with a non-covalent CDK7 inhibitor represented bystructural Formula (I), (Ia), a species thereof, or a specified formthereof, as shown and described further below. The treatment methodsinclude a step of administering such a non-covalent CDK7 inhibitor to anidentified patient, whose response can be, for example, significanttumor growth inhibition (TGI; e.g., more than about 80-90% TGI and/orcontinued tumor suppression for a period of time after cessation oftreatment). Thus, the present invention encompasses methods in which apatient is only diagnosed as being a good candidate for treatment (i.e.,identified for treatment), methods in which a patient who has beendetermined to be a good candidate for treatment (e.g., previouslyidentified) is treated, and methods requiring that a patient be bothdiagnosed and treated as described herein.

The diagnostic methods that identify a patient for treatment include astep of analyzing one or more of the biomarkers described herein in abiological sample obtained from the patient by determining, havingdetermined, or receiving information concerning the state of thebiomarker. In various embodiments, the biomarker is analyzed todetermine: whether it is present and/or in what amount (e.g., analyzedfor a genetic deletion or amplification (e.g., copy number variation(CNV)); its location (e.g., chromosomal translocation); its sequence(i.e., the analysis can include determining whether the gene is presentin wild type form or includes a mutation); whether it includes anepigenetic modification (e.g., histone and/or DNA methylation or histoneacetylation); whether it is associated with a super-enhancer (SE) or aSE of a certain strength; its level of expression (as evidenced by, forexample, the level of transcribed RNA (e.g., primary RNA or mRNA));and/or whether a protein encoded by the biomarker gene has an aberrantlevel of expression or activity (in case of doubt, a protein encoded bya biomarker gene described herein can also serve as the biomarker). Thestate of a biomarker can be assessed by examining any one or more of thefeatures just listed, and when we refer to “analyzing a/the biomarker,”we mean analyzing one or more of these features (i.e., sequence, copynumber, association with a SE, a level of RNA expression, and so forth,as provided above). For example, when we refer to analyzing thebiomarker RB1, we mean analyzing or determining whether an RB1 gene is,for example, absent in a biological sample, contains a mutation (e.g., amutation predisposing a patient to cancer), is translocated, has a CNV(copy number alteration (CNA)), bears an epigenetic modification, isassociated with a super-enhancer (SE), is overexpressed orunder-expressed (as evidenced by, for example, its level of RNA (e.g.,primary RNA or mRNA), and/or encodes a protein with a level ofexpression or activity that is above or below a predetermined thresholdlevel. As this implies, each feature analyzed can be determined to beequal to or above a pre-determined threshold level or equal to or belowa pre-determined threshold level, as described further below. Morespecifically, in the methods of the present invention, one can analyze abiomarker selected from the genes BRAF, c-myc (also known as MYC), CDK1,CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, ESR-1, FGFR1,PIK3CA, or certain genes encoding an E2F pathway member (E2F1, E2F2,E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3, CCNE1, orCCNE2; see also the Table below), or the proteins encoded thereby, bydetermining, having determined, and/or receiving information that thestate of such a biomarker, as evidenced by a feature just described(e.g., RNA level) is equal to or above (e.g., above) a pre-determinedthreshold level. Alternatively, or in addition, one can analyze abiomarker selected from the genes BCL2-like 1, CDK7, CDK9, CDKN2A, andRB (also known as RB1 or another E2F pathway member, such as RBL1, RBL2,CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C, and FBXW7), orthe proteins encoded thereby, by determining, having determined, and/orreceiving information that the state of such biomarker is equal to orbelow (e.g., below) a pre-determined threshold level. The proteinsencoded by the genes just listed as useful biomarkers in the presentmethods are known in the art. For example, BRAF encodes B-Raf; c-mycencodes MYC, CCNE1 encodes cyclin E1 (see Koff et al., Cell66:1217-1228, 1991); FGFR1 encodes FGFR1, a cell surface membranereceptor with tyrosine kinase activity; RB encodes pRB, which binds tothe activator domain of activator E2F; BCL2-like 1 encodes BCL-xL, atransmembrane protein in mitochondria; CDK7 encodes CDK7; CDK9 encodesCDK9; PIK3CA encodes the p110α protein (a catalytic subunit of the classI PI3-kinases), and CDKN2A encodes p16 and p14arf. Aliases, chromosomallocations, splice variants, and homologs of the genes and proteinsdescribed herein as biomarkers, in Homo sapiens and species other thanHomo sapiens, are also known.

The treatment methods of the invention and corresponding “uses” includeadministering, or the use of, a compound of Formula (I), any of whichmay be included in a pharmaceutically acceptable composition andadministered, e.g., by a route and regimen described herein, to apatient identified as described herein. Compounds useful in the presentmethods have structural Formula (I):

or a pharmaceutically acceptable salt, solvate, stereoisomer or mixtureof stereoisomers, tautomer, or isotopic form thereof, wherein R¹ ismethyl or ethyl; R² is methyl or ethyl; R³ is 5-methylpiperidin-3-yl,5,5-dimethylpiperidin-3-yl, 6-methylpiperdin-3-yl, or6,6-dimethylpiperidin-3-yl, wherein one or more hydrogen atoms in R³ isoptionally replaced by deuterium; and R⁴ is —CF₃ or chloro. Morespecifically, in a compound of Formula (I) or in the pharmaceuticallyacceptable salt, solvate, stereoisomer or mixture of stereoisomers,tautomer, isotopic form, or other specified form thereof (i) IV ismethyl and R² is methyl or (ii) R¹ is methyl and R² is ethyl. In otherembodiments, R¹ is ethyl and R² is ethyl. In some aspects of any one ofthese embodiments, R⁴ is —CF₃. In other aspects of any one of theseembodiments, R⁴ is chloro. In various aspects of any of the precedingembodiments, R³ is 5-methylpiperidin-3-yl, R³ is5,5-dimethylpiperidin-3-yl, R³ is 6-methyl-piperdin-3-yl, or R³ is6,6-dimethylpiperidin-3-yl, wherein one or more hydrogen atoms in R³ isoptionally replaced by deuterium. A compound of Formula (I) can havestructural Formula (Ia):

and the invention encompasses pharmaceutically acceptable salts,solvates (e.g., hydrates), tautomers, isotopic forms, or other specifiedforms of a compound of Formula (Ia), wherein R³ is

More specifically, in a compound of Formula (Ia) or a pharmaceuticallyacceptable salt, solvate, tautomer, isotopic form, or other specifiedform thereof (i) R¹ is methyl and R² is methyl or (ii) R¹ is methyl andR² is ethyl. In other embodiments, R¹ is ethyl and R² is ethyl. In someembodiments, in a compound of Formula (Ia) or a specified form thereof,R⁴ is —CF₃. In other embodiments, in a compound of Formula (Ia) or aspecified form thereof, R⁴ is chloro. In some embodiments, a compound ofFormula (I) or (Ia) is:

and the invention encompasses methods and the use of pharmaceuticallyacceptable salts, solvates (e.g., hydrates), tautomers, isotopic formsor other specified forms of any one of the three foregoing compounds. Inone embodiment, the compound is

or a pharmaceutically acceptable salt thereof. The invention alsoencompasses solvates (e.g., hydrates), tautomers, isotopic forms orother specified forms of the foregoing compound. In isotopic forms, oneor more hydrogen atoms in R³ is replaced with deuterium. In otherembodiments, none of the hydrogen atoms of a compound (e.g., none of thehydrogen atoms in R³) are replaced with deuterium. Any compound ofFormula (I), (Ia), or a species thereof can be of a “specified form,” bywhich we mean a salt, solvate (e.g., hydrate), stereoisomer (or mixturethereof), tautomer, or isotopic form of a compound of Formula (I), (Ia),or a species thereof. Also within the meaning of “specified form” areforms of a compound that manifest a combination of the attributes,features, or properties of a salt, solvate, stereoisomer, tautomer, orisotopic form. For example, the methods and uses of the invention can becarried out with a salt that has been solvated (e.g., a hydrated) or asalt of a stereoisomer, tautomer, or isotopic form of a compound ofFormula I, I(a), or a species thereof; with a solvate (e.g., hydrate)containing a salt, stereoisomer, tautomer, or isotopic form of acompound of Formula I, I(a), or a species thereof; with a stereoisomerof a compound of Formula I, I(a), or a species thereof that is in theform of a salt or solvate (e.g., hydrate) or is a tautomer or isotopicform of a compound of Formula I, I(a), or a species thereof, with atautomer of a compound of Formula I, I(a), or a species thereof that isin the form of a salt or solvate (e.g., hydrate) or that is astereoisomer or isotopic form of a compound of Formula I, I(a), or aspecies thereof; or with an isotopic form of a compound of Formula I,I(a), or a species thereof that is a salt, solvate (e.g., hydrate),stereoisomer, or tautomer of a compound of Formula I, I(a), or a speciesthereof. Any of these specified forms can be pharmaceutically acceptableand/or contained within a pharmaceutically acceptable composition (e.g.,formulated for oral administration) for use in a method describedherein.

Accordingly, the invention features treatment methods including a stepof administering a compound of structural Formula (I), or apharmaceutically acceptable salt, solvate, stereoisomer or mixture ofstereoisomers, tautomer, or isotopic form thereof, optionally within apharmaceutical composition, wherein R′, R², R³, and R⁴ are as definedherein, in treating cancer in a selected patient, wherein the patienthas been determined to have a cancer in which: (a) a gene selected fromRB1, RBL1, RBL2, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C,and FBWX7 is mutated, is genetically deleted, contains an epigeneticalteration, is translocated, is transcribed at a level equal to or belowa pre-determined threshold, or encodes a protein that is translated at alevel equal to or below a pre-determined threshold or has decreasedactivity relative to a reference standard; (b) a gene selected fromE2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CDK1, CDK2, CDK4, CDK6,CCNA1, CCNB1, CCND1, CCND2, CCND3, CCNE1, CCNE2, and BRAF is mutated, isgenetically gained or amplified, contains an epigenetic alteration, istranslocated, transcribed at a level equal to or above a pre-determinedthreshold, or encodes a protein that is translated at a level equal toor above a pre-determined threshold or has increased activity relativeto a reference standard; or (c) the gene Bcl2-like 1 is mutated,contains an epigenetic alteration, is translocated, is transcribed at alevel equal to or below a pre-determined threshold, or encodes a BCL-xLprotein that is translated at a level equal to or below a pre-determinedthreshold or has decreased activity relative to a reference standard. Inany embodiment of this method, the cancer is a blood cancer, preferablyan acute myeloid leukemia (AML), a breast cancer, preferably a triplenegative breast cancer (TNBC) or a hormone receptor positive (HR+)breast cancer, an osteosarcoma or Ewing's sarcoma, fallopian tubecancer, a GI tract cancer, preferably colorectal cancer, a glioma, alung cancer, preferably small cell or non-small cell lung cancer,melanoma, an ovarian cancer, preferably a high grade serous ovariancancer, epithelial ovarian cancer, or clear cell ovarian cancer, apancreatic cancer, a primary peritoneal cancer, prostate cancer,retinoblastoma, or a squamous cell cancer of the head or neck. Forexample, the patient may have such a cancer and can be treated asdescribed herein when it has been determined that, in a biologicalsample obtained from the patient, Bcl2-like 1 is mutated, contains anepigenetic alteration, is translocated, is transcribed at a level equalto or below a pre-determined threshold, or encodes a BCL-xL protein thatis translated at a level equal to or below a pre-determined threshold orhas decreased activity relative to a reference standard, preferablywherein a level of Bcl2-like 1 mRNA in the cancer is equal to or belowthe pre-determined threshold level. Further, such a patient can be onewho has undergone, is presently undergoing, or is prescribed treatmentwith a Bcl-2 inhibitor, as known in the art and/or described herein. Insome embodiments, the Bcl-2 inhibitor is venetoclax and the patient hasa breast cancer (e.g., TNBC); a blood cancer (e.g., AML); an ovariancancer (e.g., HGSOC); or a lung cancer (e.g., SCLC or NSCLC). In otherembodiments, the patient may have such a cancer and can be treated asdescribed herein when it has been determined that, in a biologicalsample obtained from the patient: (a) RB1 or CDKN2A is mutated, containsan epigenetic alteration, is translocated, is transcribed at a levelequal to or below a pre-determined threshold, or encodes a protein thatis translated at a level equal to or below a pre-determined threshold orhas decreased activity relative to a reference standard, preferablywherein RB1 or CDKN2A mRNA, preferably RB1 mRNA, is equal to or belowthe pre-determined threshold; and/or (b) CDK6, CCND2, or CCNE1 ismutated, has a copy number alteration, contains an epigeneticalteration, is translocated, transcribed at a level equal to or above apre-determined threshold, or encodes a protein that is translated at alevel equal to or above a pre-determined threshold or has increasedactivity relative to a reference standard, preferably wherein CDK6,CCND2, or CCNE1 mRNA, preferably CCNE1 mRNA, is equal to or above apre-determined threshold level. Such a patient can be one who hasundergone, is presently undergoing, or is prescribed treatment with aselective estrogen receptor modulator (SERM; e.g., tamoxifen,raloxifene, or toremifene), a selective estrogen receptor degrader(SERD; e.g., fulvestrant), a PARP inhibitor (e.g., olaparib orniraparib); or a platinum-based therapeutic agent (e.g., cisplatin,oxaliplatin, nedaplatin, carboplatin, phenanthriplatin, picoplatin,satraplatin (JM216). More specifically, the patient treated with a SERMor SERD may have an HR+ breast cancer; the patient treated with a PARPinhibitor may have a TNBC or a Her2⁺/ER⁻/PR⁻ breast cancer, fallopiantube cancer, glioma, ovarian cancer (e.g., an epithelial ovariancancer), or primary peritoneal cancer; and the patient treated with aplatinum-based therapeutic agent may have an ovarian cancer.

In any of the present methods where a compound of Formula (I), (Ia), aspecies thereof or a specified form thereof is used or administered,optionally within a pharmaceutical composition, the patient can be onewho has undergone, is presently undergoing, or is prescribed treatmentwith a BET inhibitor such as ABBV-075, BAY-299, BAY-1238097, BMS-986158,CPI-0610, CPI-203, FT-1101, GS-5829, GSK-2820151, GSK-525762, I-BET151,I-BET762, INCB054329, JQ1, MS436, OTX015, PFI-1, PLX51107, RVX2135,TEN-010, ZEN-3694, or a compound disclosed in U.S. application Ser. No.12/810,564; with a CDK4/6 inhibitor such as BPI-1178, G1T38,palbociclib, ribociclib, ON 123300, trilaciclib, or abemaciclib,preferably palbociclib; with a FLT3 inhibitor such as CDX-301, CG′806,CT053PTSA, crenolanib (e.g., crenolanib besylate), ENMD-2076,FF-10101-01, FLYSYN, gilteritinib (ASP2215), HM43239, lestautinib,ponatinib, NMS-088, sorafenib, sunitinib, pacritinib,pexidartinib/PLX3397, quizartinib, midostaurin, SEL24, SKI-G-801, orSKLB1028, preferably crenolanib, gilteritinib, or midostaurin; or with aMEK inhibitor such as trametinib, cobimetinib, or binimetinib. Morespecifically, a patient who has undergone, is presently undergoing, oris prescribed treatment: with a CDK4/6 inhibitor has a breast cancer,preferably a TNBC or an estrogen receptor-positive (ER⁺) breast cancer,a pancreatic cancer, or a squamous cell cancer of the head or neck; witha FLT3 inhibitor has a blood cancer, preferably AML; with a BETinhibitor has a breast cancer, preferably TNBC, a blood cancer,preferably AML, Ewing's sarcoma, or an osteosarcoma.

In any of the present methods where compound of Formula (I), (Ia), aspecies thereof or a specified form thereof is used or administered,optionally within a pharmaceutical composition, the patient can be onewho has undergone, is presently undergoing, or is prescribed treatmentwith a Bcl-2 inhibitor such as APG-1252, APG-2575, BP1002(prexigebersen), the antisense oligonucleotide known as oblimersen(G3139), S55746/BCL201, or venetoclax; a CDK9 inhibitor such asalvocidib/DSP-2033/flavopiridol, AT7519, AZD5576, BAY1251152,BAY1143572, CYC065, nanoflavopiridol, NVP2, seliciclib (CYC202), TG02,TP-1287, VS2-370 or voruciclib (formerly P1446A-05); a hormone receptor(e.g., estrogen receptor) degradation agent, such as fulvestrant; a Flt3(FMS-like tyrosine kinase 3) inhibitor such as CDX-301, CG′806,CT053PTSA, crenolanib (e.g., crenolanib besylate), ENMD-2076,FF-10101-01, FLYSYN, gilteritinib (ASP2215), HM43239, lestautinib,ponatinib, NMS-088, sorafenib, sunitinib, pacritinib,pexidartinib/PLX3397, quizartinib, midostaurin, SEL24, SKI-G-801, orSKLB1028; a PARP inhibitor such as olaparib, rucaparib, talazoparib,veliparib (ABT-888), or niraparib; a BET inhibitor such as ABBV-075,BAY-299, BAY-1238097, BMS-986158, CPI-0610, CPI-203, FT-1101, GS-5829,GSK-2820151, GSK-525762, I-BET151, I-BET762, INCB054329, JQ1, MS436,OTX015, PFI-1, PLX51107, RVX2135, TEN-010, ZEN-3694, or a compounddisclosed in U.S. application Ser. No. 12/810,564 (now U.S. Pat. No.8,476,260); a platinum-based therapeutic agent such as cisplatin,oxaliplatin, nedaplatin, carboplatin, phenanthriplatin, picoplatin,satraplatin (JM216), or triplatin tetranitrate; a CDK4/6 inhibitor suchas BPI-1178, G1T38, palbociclib, ribociclib, ON 123300, trilaciclib, orabemaciclib; a MEK inhibitor such as trametinib; or a phosphoinositide3-kinase (PI3 kinase) inhibitor, optionally of Class I (e.g., Class IA)and/or optionally directed against a specific PI3K isoform, such asidelalisib, copanlisib, duvelisib, or alpelisib; or capecitabine. Morespecifically, the second agent is selected from a Bcl-2 inhibitor suchas venetoclax, a PARP inhibitor such as olaparib or niraparib, aplatinum-based anti-cancer agent such as carboplatin or oxaliplatin, ataxane such as paclitaxel, a CDK4/6 inhibitor such as palbociclib,ribociclib, abemaciclib, or trilaciclib, a selective estrogen receptormodulator such as tamoxifen, and a selective estrogen receptor degradersuch as fulvestrant.

The invention also features kits that include a compound of Formula I,I(a), a species thereof, or a specified form thereof, and instructionalmaterial (e.g., a product insert) that describes a suitable/identifiedpatient, methods of identifying such a patient for treatment (e.g., byany one of the diagnostic stratification methods described herein foranalyzing a biomarker), and/or instructions for administering thecompound of Formula I, I(a), a species thereof, or a specified formthereof, alone or in combination with at least one other therapeuticagent (e.g., an additional/second anti-cancer therapeutic including anyone or more of the second agents described herein). The kits of theinvention can also include the second agent (e.g., an anti-canceragent), including any one or more of the second agents described hereinand instructions for use in a population of patients identified asdescribed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table depicting the inhibitory and dissociation constantsand selectivity of the indicated compounds (three compounds of theinvention and four comparators) against CDK2, CDK7, CDK9, and CDK12.

FIG. 2 is a line graph depicting changes in tumor volume (mm³) over time(days) in a palbociclib-resistant HR+BC PDX model (ST1799), as describedin the Examples below.

FIG. 3 is a line graph depicting tumor volume (mm³) over time (days) inthe palbociclib- and fulvestrant-resistant HR+BC PDX model ST941, asdescribed in the Examples below.

FIG. 4 is a panel showing three line graphs that depict changes in tumorvolume (mm³) over time (days) in PDX models of TNBC (BR5010; top), smallcell lung cancer (LU5178; middle), and ovarian cancer (OV15398; bottom).The animals were treated with Compound 101 as described in Example 10.Data obtained from vehicle-treated (control) animals is represented byfilled circles (upper traces in each graph). Data from animals modelingTNBC and given 10 mg/kg Compound 101 QD are represented in the top graphby filled squares; the dose of 5 mg/kg BID is represented by triangles.Triangles also represent data obtained from the animal models of SCLCand ovarian cancer treated with Compound 101 in the middle and bottomgraphs.

FIG. 5 is a panel of line graphs showing tumor growth in the PDX modelsindicated and corresponding isobolograms, each generated as described inExample 11. Compound 101 was applied to cells in combination with theindicated second agents at the concentrations shown.

FIG. 6 is a panel of graphs generated from data collected in theCompound 101-treated PDX models described in Example 12. Black lineswith squares represent vehicle-treated animals. Gray lines representCompound 101-treated animals. Error bars are SEM. BID=twice daily;CNV=copy number variation; MPK=mg per kg body weight; PO=oral; QD=oncedaily; RB=retinoblastoma; SCLC=small cell lung cancer; TNBC=triplenegative breast cancer. Vertical dotted lines mark the last day oftreatment.

FIG. 7 is a Table summarizing the TGI values and genetic status of the12 PDX models studied as described in Example 12. Models in the tableare sorted based on highest to lowest response at end of study. BID,CNV, RB, SCLC, and TNBC are as defined for FIG. 6 and elsewhere herein.In case of doubt, CCNE1=the gene encoding cyclin E1;CDKN2A=cyclin-dependent kinase inhibitor 2A, EoS=end of study, EoT=endof treatment, HGSOC=high-grade serous ovarian cancer, OVA=ovariancancer, and TGI=tumor growth inhibition. For the LU5210 model, tissuewas not available for confirmation of RB pathway genetics.

DETAILED DESCRIPTION

Despite the efficacy of compounds of Formula (I), we believe that suchefficacy will be higher in patients that have certain genetic signatures(i.e., biomarkers in a particular state, which can be analyzed asdescribed herein). Moreover, we believe the efficacy of compounds ofFormula (I) may be enhanced when combined with other anti-cancertherapies in newly diagnosed and refractory cancer patients identifiedas described herein.

The following definitions apply to the compositions, methods, and usesdescribed herein unless the context clearly indicates otherwise, and itis to be understood that the claims may be amended to include languagewithin a definition as needed or desired. Moreover, the definitionsapply to linguistic and grammatical variants of the defined terms (e.g.,the singular and plural forms of a term), and some linguistic variantsare particularly mentioned below (e.g., “administration” and“administering”). The chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75th Ed. Additionally, general principles oforganic chemistry are well established and one of ordinary skill in theart can consult, if desired, Organic Chemistry by Thomas Sorrell,University Science Books, Sausalito, 1999; Smith and March, March'sAdvanced Organic Chemistry, 5^(th) Edition, John Wiley & Sons, Inc., NewYork, 2001; Larock, Comprehensive Organic Transformations, VCHPublishers, Inc., New York, 1989; and Carruthers, Some Modern Methods ofOrganic Synthesis, 3^(rd) Edition, Cambridge University Press,Cambridge, 1987.

The term “about,” when used in reference to a value, signifies any valueor range of values that is plus-or-minus 10% of the stated value (e.g.,within plus-or-minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of thestated value). For example, a dose of about 10 mg means any dose as lowas 10% less than 10 mg (9 mg), any dose as high as 10% more than 10 mg(11 mg), and any dose or dosage range therebetween (e.g., 9-11 mg;9.1-10.9 mg; 9.2-10.8 mg; and so on). As another example, a prevalencerank in a population of about 80% means a prevalence rank of 72-88%(e.g., 79.2-80.8%). In case of doubt, “about X” can be “X” (e.g., about80% can be 80%). Where a stated value cannot be exceeded (e.g., 100%),“about” signifies any value or range of values that is up to andincluding 10% less than the stated value (e.g., a purity of about 100%means 90%-100% pure (e.g., 95%-100% pure, 96%-100% pure, 97%-100% pureetc. . . . )). In the event an instrument or technique measuring a valuehas a margin of error greater than 10%, a given value will be about thesame as a stated value when they are both within the margin of error forthat instrument or technique.

The term “administration” and variants thereof, such as “administering,”refer to the administration of a compound described herein (e.g., acompound of Formula (I), (Ia), a species thereof or a specified formthereof (e.g., a pharmaceutically acceptable salt of a compound ofFormula (I), (Ia), or a species thereof), or an additional/secondagent), or a composition containing the compound to a subject (e.g., ahuman patient) or system (e.g., a cell- or tissue-based system that ismaintained ex vivo); as a result of the administration, the compound orcomposition containing the compound (e.g., a pharmaceutical composition)is introduced to the subject or system. In addition to compositions ofthe invention and second agents useful in combination therapies, itemsused as positive controls, negative controls, and placebos, any of whichcan also be a compound, can also be “administered.” One of ordinaryskill in the art will be aware of a variety of routes that can, inappropriate circumstances, be utilized for administration to a subjector system. For example, the route of administration can be oral (i.e.,by swallowing a pharmaceutical composition) or may be parenteral. Morespecifically, the route of administration can be bronchial (e.g., bybronchial instillation), by mouth (i.e., oral), dermal (which may be orcomprise topical application to the dermis or intradermal, interdermal,or transdermal administration), intragastric or enteral (i.e., directlyto the stomach or intestine, respectively), intramedullary,intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral,intravenous (or intra-arterial), intraventricular, by application to orinjection into a specific organ (e.g., intrahepatic), mucosal (e.g.,buccal, rectal, sublingual, or vaginal), subcutaneous, tracheal (e.g.,by intratracheal instillation), or ocular (e.g., topical,subconjunctival, or intravitreal). Administration can involveintermittent dosing (i.e., doses separated by various times) and/orperiodic dosing (i.e., doses separated by a common period of time (e.g.,every so many hours, daily (e.g., once daily oral dosing), weekly, twiceper week, etc.)). In other embodiments, administration may involvecontinuous dosing (e.g., perfusion) for a selected time (e.g., about 1-2hours).

Two events, two entities, or an event and an entity are “associated”with one another if one or more features of the first (e.g., itspresence, level and/or form) are correlated with a feature of thesecond. For example, a first entity (e.g., an enzyme (e.g., CDK7)), geneexpression profile, genetic signature (i.e., a single or combined groupof genes in a cell with a uniquely characteristic pattern of geneexpression), metabolite, or event (e.g., myeloid infiltration)) isassociated with an event (e.g., the onset or progression of a particulardisease), if its presence, level and/or form correlates with theincidence of, severity of, and/or susceptibility to the disease (e.g., acancer disclosed herein). The biomarkers described herein are associatedwith an identified patient in the manner described herein (e.g., byvirtue of their level of expression) and, depending on their status, canalso be associated with a clinical outcome (e.g., a better prognosisbased on an increased likelihood that a treatment regimen describedherein will be more successful as evidenced by, e.g., TGI, preferablybeyond the cessation of treatment). Associations are typically assessedacross a relevant population. Two or more entities are physically“associated” with one another if they interact, directly or indirectly,so that they are and/or remain in physical proximity with one another ina given circumstance (e.g., within a cell maintained under physiologicalconditions (e.g., within cell culture) or within a pharmaceuticalcomposition). Entities that are physically associated with one anothercan be covalently linked to one another or non-covalently associated by,for example, hydrogen bonds, van der Waals forces, hydrophobicinteractions, magnetism, or combinations thereof. A compound of Formula(I), (Ia), a species thereof, or a specified form thereof (e.g., apharmaceutically acceptable salt) can non-covalently associate withCDK7.

The term “biological sample” refers to a sample obtained or derived froma biological source of interest (e.g., a tissue or organism (e.g., ananimal or human patient) or cell culture). For example, a biologicalsample can be a sample obtained from an individual (e.g., a patient oran animal model) suffering from a disease (or, in the case of an animalmodel, a simulation of that disease in a human patient) to be diagnosedand/or treated by the methods of this invention or from an individualserving in the capacity of a reference or control (or whose samplecontributes to a reference standard or control population). Thebiological sample can contain a biological cell, tissue or fluid or anycombination thereof. For example, a biological sample can be or caninclude ascites; blood; blood cells; a bodily fluid, any of which mayinclude or exclude cells (e.g., tumor cells (e.g., circulating tumorcells (CTCs) found in at least blood or lymph vessels)) or circulatingtumor DNA (ctDNA); bone marrow or a component thereof (e.g.,hematopoietic cells, marrow adipose tissue, or stromal cells);cerebrospinal fluid (CSF); feces; flexural fluid; free-floating nucleicacids (e.g., circulating tumor DNA); gynecological fluids; hair; immuneinfiltrates; lymph; peritoneal fluid; plasma; saliva; skin or acomponent part thereof (e.g., a hair follicle); sputum;surgically-obtained specimens; tissue scraped or swabbed from the skinor a mucus membrane (e.g., in the nose, mouth, or vagina); tissue orfine needle biopsy samples; urine; washings or lavages such as a ductallavage or broncheoalveolar lavage; or other body fluids, tissues,secretions, and/or excretions. Samples of, or samples obtained from, abodily fluid (e.g., blood, CSF, lymph, plasma, or urine) may includetumor cells (e.g., CTCs) and/or free-floating or cell-free nucleic acidsof the tumor. Cells (e.g., cancer cells) within the sample may have beenobtained from an individual patient for whom a treatment is intended.Samples used in the form in which they were obtained may be referred toas “primary” samples, and samples that have been further manipulated(e.g., by removing one or more components of the sample) may be referredto as “secondary” or “processed” samples. Such processed samples maycontain or be enriched for a particular cell type (e.g., aCDK7-expressing cell, which may be a tumor cell), cellular component(e.g., a membrane fraction), or cellular material (e.g., one or morecellular proteins, including CDK7, DNA, or RNA (e.g., mRNA), which mayencode CDK7 and may be subjected to amplification). As used herein, theterm “biomarker” refers to an entity whose state correlates with aparticular biological event so that it is considered to be a “marker”for that event (e.g., the presence of a particular cancer and itssusceptibility to a compound of Formula (I), (Ia), a species thereof, ora specified form thereof). A biomarker can be analyzed at the nucleicacid or protein level; at the nucleic acid level, one can analyze thepresence (e.g., copy number alterations (CNAs)), absence, or chromosomallocation of a gene in wild type or mutant form, epigenetic alterations(in, e.g., methylation), its association with a super-enhancer, and/orits level of expression (as evidenced, for example, by primary RNAtranscript or mRNA levels). At the protein level, one can analyze thelevel of expression and/or activity of a protein encoded by a biomarkergene. A biomarker may indicate a therapeutic outcome or likelihood(e.g., increased likelihood) thereof. Thus, a biomarker can bepredictive or prognostic and is therefore useful in methods ofidentifying or treating a patient as described herein.

The term “cancer” refers to a disease in which biological cells exhibitan aberrant growth phenotype characterized by loss of control of cellproliferation to an extent that will be detrimental to a patient havingthe disease. A cancer can be classified by the type of tissue in whichit originated (histological type) and/or by the primary site in the bodyin which the cancer first developed. Based on histological type, cancersare generally grouped into six major categories: carcinomas; sarcomas;myelomas; leukemias; lymphomas; and mixed types. A cancer treated asdescribed herein may be of any one of these types and may comprise cellsthat are precancerous (e.g., benign), malignant, pre-metastatic,metastatic, and/or non-metastatic. A patient who has a malignancy ormalignant lesion has a cancer. The present disclosure specificallyidentifies certain cancers to which its teachings may be particularlyrelevant, and one or more of these cancers may be characterized by asolid tumor or by a hematologic tumor, which may also be known as ablood cancer (e.g., of a type described herein). Although not allcancers manifest as solid tumors, we may use the terms “cancer cell” and“tumor cell” interchangeably to refer to any malignant cell.

The term “combination therapy” refers to those situations in which asubject is exposed to two or more therapeutic regimens (e.g., two ormore therapeutic agents) to treat a single disease (e.g., a cancer). Thetwo or more regimens/agents may be administered simultaneously orsequentially. When administered simultaneously, a dose of the firstagent and a dose of the second agent are administered at about the sametime, such that both agents exert an effect on the patient at the sametime or, if the first agent is faster- or slower-acting than the secondagent, during an overlapping period of time. When administeredsequentially, the doses of the first and second agents are separated intime, such that they may or may not exert an effect on the patient atthe same time. For example, the first and second agents may be givenwithin the same hour or same day, in which case the first agent wouldlikely still be active when the second is administered. Alternatively, amuch longer period of time may elapse between administration of thefirst and second agents, such that the first agent is no longer activewhen the second is administered (e.g., all doses of a first regimen areadministered prior to administration of any dose(s) of a second regimenby the same or a different route of administration, as may occur intreating a refractory cancer). For clarity, combination therapy does notrequire that two agents be administered together in a single compositionor at the same time, although in some embodiments, two or more agents,including a compound of Formula (I), (Ia), a species thereof, or aspecified form thereof and a second agent described herein, may beadministered within the same period of time (e.g., within the same hour,day, week, or month).

The terms “cutoff” and “cutoff value” mean a value measured in an assaythat defines the dividing line between two subsets of a population(e.g., likely responders and non-responders (e.g., responders andnon-responders to a compound of Formula (I), (Ia), a species thereof, ora specified form thereof)). In some instances, values that are equal toor above the cutoff value define one subset of the population, andvalues that are lower than the cutoff value define the other subset ofthe population. In other instances, values that are equal to or belowthe cutoff value define on subset of the population, and values abovethe cutoff value define the other. As described further below, thecutoff or cutoff value can define the threshold value.

As used herein, “diagnostic information” is information that is usefulin determining whether a patient has a disease and/or in classifying(stratifying) the disease into a genotypic or phenotypic category or anycategory having significance with regard to the prognosis of the diseaseor its likely response to treatment (either treatment in general or anyparticular treatment described herein). Similarly, “diagnosis” refers toobtaining or providing any type of diagnostic information, including,but not limited to, whether a patient is likely to have or develop adisease; whether that disease has or is likely to reach a certain stateor stage or to exhibit a particular characteristic (e.g., resistance toa therapeutic agent); information related to the nature orclassification of a tumor; information related to prognosis (which mayalso concern resistance); and/or information useful in selecting anappropriate treatment (e.g., selecting a compound of Formula (I), (Ia),a species thereof, or a specified form thereof for a patient identifiedas having a cancer that is likely to respond to such an inhibitor orother treatment). A patient classified (stratified) according to amethod described herein and selected for treatment with a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof islikely to respond well to the treatment, meaning that such a patient ismore likely to be successfully treated than a patient with the same typeof cancer who has not been so identified and is not in the same strata.Available treatments include therapeutic agents and other treatmentmodalities such as surgery, radiation, etc., and selecting anappropriate treatment encompasses the choice of withholding a particulartherapeutic agent; the choice of a dosing regimen; and the choice ofemploying a combination therapy. Diagnostic information can be used tostratify patients and is thus useful in identifying and classifying agiven patient according to, for example, biomarker status. Obtainingdiagnostic information can constitute a step in any of the patientstratification methods described herein.

One of ordinary skill in the art will appreciate that the term “dosageform” may be used to refer to a physically discrete unit of an activeagent (e.g., a therapeutic or diagnostic agent) for administration to apatient. Typically, each such unit contains a predetermined quantity ofactive agent. In some embodiments, such quantity is a unit dosage amount(or a whole fraction thereof) appropriate for administration inaccordance with a dosing regimen that has been determined to correlatewith a desired or beneficial outcome when administered to a relevantpopulation (i.e., with a therapeutic dosing regimen). Those of ordinaryskill in the art appreciate that the total amount of a therapeuticcomposition or agent administered to a particular patient is determinedby one or more attending physicians and may involve administration ofmultiple dosage forms.

One of ordinary skill in the art will appreciate that the term “dosingregimen” may be used to refer to a set of unit doses (typically morethan one) that are administered individually to a patient, separated byequal or unequal periods of time. A given therapeutic agent typicallyhas a recommended dosing regimen, which may involve one or more doses,each of which may contain the same unit dose amount or differingamounts. In some embodiments, a dosing regimen comprises a first dose ina first dose amount, followed by one or more additional doses in asecond dose amount that is different from the first dose amount. In someembodiments, a dosing regimen is correlated with a desired or beneficialoutcome when administered across a relevant population (i.e., theregimen is a therapeutic dosing regimen).

As used herein, an “effective amount” of an agent (e.g., a chemicalcompound described herein), such as a compound of Formula (I), refers toan amount that produces or is expected to produce the desired effect forwhich it is administered. The effective amount will vary depending onfactors such as the desired biological endpoint, the pharmacokinetics ofthe compound administered, the condition being treated, the mode ofadministration, and characteristics of the patient, as discussed furtherbelow and recognized in the art. The term can be applied to therapeuticand prophylactic methods. For example, a therapeutically effectiveamount is one that reduces the incidence and/or severity of one or moresigns or symptoms of the disease. For example, in treating a cancer, aneffective amount may reduce the tumor burden, stop tumor growth, inhibitmetastasis or prolong patient survival. One of ordinary skill in the artwill appreciate that the term does not in fact require successfultreatment be achieved in any particular individual. Rather, atherapeutically effective amount is that amount that provides aparticular desired pharmacological response in a significant number ofpatients when administered to patients in need of such treatment. Insome embodiments, reference to a therapeutically effective amount may bea reference to an amount administered or an amount measured in one ormore specific tissues (e.g., a tissue affected by the disease) or fluids(e.g., blood, saliva, serum, sweat, tears, urine, etc.). Effectiveamounts may be formulated and/or administered in a single dose or in aplurality of doses, for example, as part of a dosing regimen.

As used herein, an “enhancer” is a region of genomic DNA that helpsregulate the expression of genes; enhancers have been found up to 1 Mbpaway from a gene they regulate. An enhancer may overlap, but is oftennot composed of, gene coding regions. An enhancer is often bound bytranscription factors and designated by specific histone marks.

The term “patient” refers to any organism that is or may be subjected toa diagnostic method described herein or to which a compound describedherein, or a specified form thereof, is or may be administered for,e.g., experimental, diagnostic, prophylactic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and humans; domesticated animals,such as dogs and cats; and livestock or any other animal of agriculturalor commercial value). A patient may be suffering from or susceptible to(i.e., have a higher than average risk of developing) a diseasedescribed herein and may display one or more signs or symptoms thereof.

The term “pharmaceutically acceptable,” when applied to a carrier usedto formulate a composition disclosed herein (e.g., a pharmaceuticalcomposition), means a carrier that is compatible with the otheringredients of the composition and not deleterious to a patient (e.g.,it is non-toxic in the amount required and/or administered (e.g., in aunit dosage form)).

The term “pharmaceutically acceptable,” when applied to a salt, solvate,stereoisomer, tautomer, or isotopic form of a compound described herein,refers to a salt, solvate, stereoisomer, tautomer, or isotopic form thatis, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans (e.g., patients) and lower animals(including, but not limited to, mice and rats used in laboratorystudies) without unacceptable toxicity, irritation, allergic responseand the like, and that can be used in a manner commensurate with areasonable benefit/risk ratio. Many pharmaceutically acceptable saltsare well known in the art (see, e.g., Berge et al., J. Pharm. Sci.66:1-19, 1977). Pharmaceutically acceptable salts of the compoundsdescribed herein include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid, and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid, or malonic acid or by using other methods known in theart such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, MALAT1e, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, andaryl sulfonate.

As used herein, the term “population” means some number of items (e.g.,at least 30, 40, 50, or more) sufficient to reasonably reflect thedistribution, in a larger group, of the value being measured in thepopulation. Within the context of the present invention, the populationcan be a discrete group of humans, laboratory animals, or cells lines(for example) that are identified by at least one common characteristicfor the purposes of data collection and analysis. For example, a“population of samples” refers to a plurality of samples that is largeenough to reasonably reflect the distribution of a value (e.g., a valuerelated to the state of a biomarker) in a larger group of samples. Theitems in the population may be biological samples, as described herein.For example, each sample in a population of samples may be cells of acell line or a biological sample obtained from a patient or a xenograft(e.g., a tumor grown in a mouse by implanting a tumorigenic cell line ora patient sample into the mouse). As noted, individuals within apopulation can be a discrete group identified by a commoncharacteristic, which can be the same disease (e.g., the same type ofcancer), whether the sample is obtained from living beings sufferingfrom the same type of cancer or a cell line or xenograft representingthat cancer.

The term “prevalence cutoff,” as used herein in reference to a specifiedvalue (e.g., the strength of a SE associated with a biomarker gene)means the prevalence rank that defines the dividing line between twosubsets of a population (e.g., a subset of “responders” and a subset of“non-responders,” which, as the names imply include patients who arelikely or unlikely, respectively, to experience a beneficial response toa therapeutic agent or agents). Thus, a prevalence rank that is equal toor higher (e.g., a lower percentage value) than the prevalence cutoffdefines one subset of the population; a prevalence rank that is lower(e.g., a higher percentage value) than the prevalence cutoff defines theother subset.

As used herein, the term “prevalence rank” for a specified value (e.g.,the mRNA level of a specific biomarker) means the percentage of apopulation that are equal to or greater than that specific value. Forexample, a 35% prevalence rank for the amount of mRNA of a specificbiomarker in a test cell means that 35% of the population have thatlevel of biomarker mRNA or greater than the test cell.

As used herein, the terms “prognostic information” and “predictiveinformation” are used to refer to any diagnostic information that may beused to indicate any aspect of the course of a disease or conditioneither in the absence or presence of treatment. Such information mayinclude, but is not limited to, the average life expectancy of apatient, the likelihood that a patient will survive for a given amountof time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that apatient will be cured of a disease, the likelihood that a patient'sdisease will respond to a particular therapy (wherein response may bedefined in any of a variety of ways). Diagnostic information can beprognostic or predictive.

As used herein, the term “rank ordering” means the ordering of valuesfrom highest to lowest or from lowest to highest.

As used herein, the terms “RB-E2F pathway” and “RB-E2F family” refer toa set of genes and the proteins encoded thereby, as the context willmake clear, whose expression or activity regulates the activity of theRB gene family and in turn regulates the activity of the E2F family oftranscription factors that are required for entry into and progressionthrough the cell cycle. The table below contains a list of genes in theRB-E2F family, an indication of a currently understood function of theencoded proteins and the status of these biomarkers in cancer. We usethe shorthand “activated or overexpressed” to indicate that an attributeof a gene (e.g., its copy number or level of expression) or the proteinit encodes (e.g., its level of expression or activity) is higher in somepatients with certain cancers than it is in healthy subjects. Apre-determined threshold for such activated or overexpressed RB-E2Ffamily members can be determined by comparative analysis and is a level(e.g., mRNA level, protein level, gene copy number, strength of enhancerassociated with the gene) that, when found or exceeded in a cancerpatient, identifies that patient as a candidate for treatment asdescribed herein. We use the shorthand “inactivated or underexpressed”to indicate that an attribute of a gene (e.g., its copy number, or levelof expression) or a protein it encodes (e.g., its level of expression oractivity) is lower in some patients with certain cancers than it is inhealthy subjects. A pre-determined threshold for such inactivated orunderexpressed RB-E2F family members can be determined by comparativeanalysis and is a level (e.g., mRNA level, protein level, CNV, strengthof enhancer associated with the gene) that, when unattained in a cancerpatient, identifies that patient as a candidate for treatment asdescribed herein.

Gene Function Status in Cancer E2F1 E2F family - transcriptional controlof cell cycle Activated or overexpressed entry E2F2 E2F family -transcriptional control of cell cycle Activated or overexpressed entryE2F3 E2F family - transcriptional control of cell cycle Activated oroverexpressed entry E2F4 E2F family - transcriptional control of cellcycle Activated or overexpressed entry E2F5 E2F family - transcriptionalcontrol of cell cycle Activated or overexpressed entry E2F6 E2F family -transcriptional control of cell cycle Activated or overexpressed entryE2F7 E2F family - transcriptional control of cell cycle Activated oroverexpressed entry E2F8 E2F family - transcriptional control of cellcycle Activated or overexpressed entry RB1 RB family - E2F familyinhibition Inactivated or underexpressed RBL1 RB family - E2F familyinhibition Inactivated or underexpressed RBL2 RB family - E2F familyinhibition Inactivated or underexpressed CDK4 RB family inhibitionActivated or overexpressed CDK6 RB family inhibition Activated oroverexpressed CDK2 RB family inhibition Activated or overexpressed CCND1CDK4/6 regulation Activated or overexpressed CCND2 CDK4/6 regulationActivated or overexpressed CCND3 CDK4/6 regulation Activated oroverexpressed CDKN2A CDK4/6 regulation Inactivated or underexpressedCDKN2B CDK4/6 regulation Inactivated or underexpressed CDKN2C CDK4/6regulation Inactivated or underexpressed CDKN2D CDK4/6 regulationInactivated or underexpressed CCNE1 CDK2 regulation Activated oroverexpressed CCNE2 CDK2 regulation Activated or overexpressed CDKN1ACDK2 regulation Inactivated or underexpressed CDKN1B CDK2 regulationInactivated or underexpressed CDKN1C CDK2 regulation Inactivated orunderexpressed FBXW7 CCNE regulation Inactivated or underexpressedIt will be readily apparent to one of ordinary skill in the art that forthose genes in the RB-E2F pathway that are activated or overexpressed incancer, one would select those patients that had (1) an alteration inthe DNA encoding such gene that resulted in increased expression (e.g.elevated gene copy number, mutation that led to increased activity,change in methylation that led to increased expression); (2) anepigenetic alteration associated with that gene that resulted inincreased expression (e.g. histone methylation or histone acetylationpattern that led to increased expression); or (3) an increase in thelevel of expression of mRNA or protein encoded by that gene. For thosegenes in the RB-E2F pathway that are inactivated or under-expressed incancer, one would select from those patients that had (1) an alterationin the DNA encoding that gene that resulted in decreased expression oractivity (e.g. reduced gene copy number, mutation that led to decreasedactivity or inactivity, change in methylation that led to decreasedexpression); (2) an epigenetic alteration associated with that gene thatresulted in decreased expression (e.g. histone methylation or histoneacetylation pattern that led to decreased expression); or (3) andecrease in the level of expression of mRNA or protein encoded by thatgene.

As used herein, a “reference” refers to a standard or control relativeto which a comparison is performed. For example, an agent, patient,population, sample, sequence, or value of interest is compared with areference agent, patient, population, sample, sequence or value. Thereference can be analyzed or determined substantially simultaneouslywith the analysis or determination of the item of interest or it mayconstitute a historical standard or control, determined at an earlierpoint in time and optionally embodied in a tangible medium. One ofordinary skill in the art is well trained in selecting appropriatereferences, which are typically determined or characterized underconditions that are comparable to those encountered by the item ofinterest. One of ordinary skill in the art will appreciate whensufficient similarities are present to justify reliance on and/orcomparison to a particular possible reference as a standard or control.

As used herein, a “response” to treatment is any beneficial alterationin a patient's condition that results from, or that correlates with,treatment. The alteration may be stabilization of the condition (e.g.,inhibition of deterioration that would have taken place in the absenceof the treatment), amelioration of, delay of onset of, and/or reductionin frequency of one or more signs or symptoms of the condition,improvement in the prospects for cure of the condition, greater survivaltime, and etc. A response may be a patient's response or a tumor'sresponse.

As used herein, when the term “strength” is used to refer to a portionof an enhancer or a SE, it means the area under the curve of the numberof H3K27Ac or other genomic marker reads plotted against the length ofthe genomic DNA segment analyzed. Thus, “strength” is an integration ofthe signal resulting from measuring the mark at a given base pair overthe span of the base pairs defining the region being chosen to measure.

As used herein, the term “super-enhancer” (SE) refers to a subset ofenhancers that contain a disproportionate share of histone marks and/ortranscriptional proteins relative to other enhancers in a particularcell or cell type. Genes regulated by SEs are predicted to be of highimportance to the function of a cell. SEs are typically determined byrank ordering all of the enhancers in a cell based on strength anddetermining, using available software such as ROSE(bitbucket.org/young_computation/rose), the subset of enhancers thathave significantly higher strength than the median enhancer in the cell(see, e.g., U.S. Pat. No. 9,181,580, which is hereby incorporated byreference herein in its entirety).

The terms “threshold” and “threshold level” mean a level that definesthe dividing line between two subsets of a population (e.g., respondersand non-responders). A threshold or threshold level can define aprevalence cutoff or a cutoff value.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, and/or inhibiting theprogress of a “pathological condition” (e.g., a disease, such as cancer)described herein. In some embodiments, “treatment,” “treat,” and“treating” require that signs or symptoms of the disease have developedor have been observed. In other embodiments, treatment may beadministered in the absence of signs or symptoms of the disease orcondition (e.g., in light of a history of symptoms and/or in light ofgenetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example, to delay or inhibitrecurrence.

As the invention relates to compositions and methods for diagnosing andtreating patients who have cancer, the terms “active agent,”“anti-cancer agent,” “pharmaceutical agent,” and “therapeutic agent” areused interchangeably (unless the context clearly indicates otherwise)and compounds of Formula (I), (Ia), a species thereof, or a specifiedform thereof, would be understood by one of ordinary skill in the art asactive, anti-cancer, pharmaceutical, or therapeutic agents. As noted,the treatment methods and uses encompass combination therapies/uses inwhich a compound of Formula (I), (Ia), a species thereof, or a specifiedform thereof is administered or used in combination with one or moreadditional agents (e.g., an additional anti-cancer therapeutic), asdescribed herein. In keeping with convention, in any embodimentrequiring two agents, we may refer to one as the “first” agent and tothe other as the “second” agent to underscore that the first and secondagents are distinct from one another. Where three agents are employed,we refer to the “third agent.”

As indicated, each therapeutic method and any diagnostic method thatemploys a compound of Formula (I), (Ia), a species thereof, or aspecified form thereof may also be expressed in terms of use and viceversa. For example, the invention encompasses the use of a compound orcomposition described herein for the treatment of a disease describedherein (e.g., cancer); a compound or composition for use in diagnosingand/or treating or a disease (e.g., cancer); and the use of the compoundor composition for the preparation of a medicament for treating adisease described herein (e.g., cancer).

A patient subjected to a diagnostic or therapeutic method describedherein may have a blood cancer, which may also be referred to as ahematopoietic or hematological cancer or malignancy, and any of themethods described herein can entail analyzing a biomarker describedherein in a biological sample of, e.g., blood or lymph, obtained fromthe patient. More specifically and in various embodiments, the bloodcancer can be a leukemia such as acute lymphocytic leukemia (ALL; e.g.,B cell ALL or T cell ALL), acute myelocytic leukemia (AML; e.g., B cellAML or T cell AML), chronic myelocytic leukemia (CML; e.g., B cell CMLor T cell CML), chronic lymphocytic leukemia (CLL; e.g., B cell CLL(e.g., hairy cell leukemia) or T cell CLL), chronic neutrophilicleukemia (CNL), or chronic myelomonocytic leukemia (CMML). The bloodcancer can also be a lymphoma such as Hodgkin lymphoma (HL; e.g., B cellHL or T cell HL), non-Hodgkin lymphoma (NHL, which can be deemedaggressive; e.g., B cell NHL or T cell NHL), follicular lymphoma (FL),chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL),mantle cell lymphoma (MCL), a marginal zone lymphoma (MZL), such as a Bcell lymphoma (e.g., splenic marginal zone B cell lymphoma), primarymediastinal B cell lymphoma (e.g., splenic marginal zone B celllymphoma), primary mediastinal B cell lymphoma, Burkitt lymphoma (BL),lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia),immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma,or primary central nervous system (CNS) lymphoma. The B cell NHL can bediffuse large cell lymphoma (DLCL; e.g., diffuse large B cell lymphoma(DLBCL; e.g., germinal center B cell-like (GCB) DLBCL or activatedB-cell like (ABC) DLBCL)), and the T cell NHL can be precursor Tlymphoblastic lymphoma or a peripheral T cell lymphoma (PTCL). In turn,the PTCL can be a cutaneous T cell lymphoma (CTCL) such as mycosisfungoides or Sezary syndrome, angioimmunoblastic T cell lymphoma,extranodal natural killer T cell lymphoma, enteropathy type T celllymphoma, subcutaneous anniculitis-like T cell lymphoma, or anaplasticlarge cell lymphoma.

In other embodiments, the cancer is characterized by a solid tumorconsidered to be either of its primary location or metastatic. Forexample, in various embodiments, the cancer or tumor treated orprevented as described herein is an acoustic neuroma; adenocarcinoma;adrenal gland cancer; anal cancer; angiosarcoma (e.g.,lymphangiosarcoma, lymphangio-endotheliosarcoma, hemangiosarcoma);appendix cancer; benign monoclonal gammopathy (also known as monoclonalgammopathy of unknown significance (MGUS); biliary cancer (e.g.,cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast; any of which may be present insubjects having a particular profile, such as an HR+(ER+ or PR+), HER2+,HR− (having neither estrogen nor progesterone receptors), a triplenegative breast cancer (TNBC; ER−/PR−/HER2−), or a triple-positivebreast cancer (ER+/PR+/HER2+); a brain cancer (e.g., meningioma,glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma),medulloblastoma); bronchus cancer; carcinoid tumor, which may be benign;cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma;chordoma; craniopharyngioma; a cancer present in the large intestine,such as colorectal cancer (CRC, e.g., colon cancer, rectal cancer, orcolorectal adenocarcinoma); connective tissue cancer; epithelialcarcinoma; ependymoma; endothelio-sarcoma (e.g., Kaposi's sarcoma ormultiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g.,uterine cancer, uterine sarcoma); esophageal cancer (e.g.,adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing'ssarcoma (or other pediatric sarcoma, such as embryonal rhabdomyosarcomaor alveolar rhabdomyosarcoma); eye cancer (e.g., intraocular melanoma,retinoblastoma); familiar hypereosinophilia; gallbladder cancer; gastriccancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor(GIST); germ cell cancer; head and neck cancer (e.g., head and necksquamous cell carcinoma, oral cancer (e.g., oral squamous cellcarcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer,nasopharyngeal cancer, oropharyngeal cancer)); hypopharynx cancer;inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidneycancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma);liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma);lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer(SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma, squamouscell carcinoma, or large cell carcinoma of the lung); leiomyosarcoma(LMS); mastocytosis (e.g., systemic mastocytosis); mouth cancer; musclecancer; myelodys-plastic syndrome (MDS); mesothelioma;myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, hypereosinophilicsyndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g.,gastroentero-pancreatic neuroendocrine tumor (GEP-NET), carcinoidtumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g.,cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma,HGSOC, LGSOC, epithelial ovarian cancer (e.g., ovarian clear cellcarcinoma or mucinous carcinoa), sex cord stromal tumors (granulosacell), and endometroid tumors); papillary adenocarcinoma; pancreaticcancer (whether an exocrine tumor (e.g., pancreatic adenocarcinoma,pancreatic ductal adenocarcinoma (PDAC)), intraductal papillary mucinousneoplasm (IPMN), or a neuroendocrine tumor (e.g., PNETs or islet celltumors); penile cancer (e.g., Paget's disease of the penis and scrotum);pinealoma; primary peritoneal cancer, primitive neuroectodermal tumor(PNT); plasma cell neoplasia; paraneoplastic syndromes; prostate cancer,which may be castration-resistant (e.g., prostate adenocarcinoma);rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamouscell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cellcarcinoma (BCC)); small bowel or small intestine cancer; soft tissuesarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma,malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma,fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; sweat glandcarcinoma; synovioma; testicular cancer (e.g., seminoma, testicularembryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of thethyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer);urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget'sdisease of the vulva). We use the term “gastrointestinal (GI) tractcancer” to refer to a cancer present anywhere in the GI tract, includingcancers of the mouth, throat, esophagus, stomach, large or smallintestine, rectum, and anus. As noted above, the cancer can be aneuroendocrine cancer, and such tumors can be treated as describedherein regardless of the organ in which they present. A biomarkerdescribed herein can be analyzed in a biological sample containing tumorcells or ctDNA of any of the cancer types just listed. Further, apatient identified by analyzing a biomarker as described herein can be“newly diagnosed” and therefor previously unexposed to a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof and,similarly, previously unexposed to a second agent as described herein.We may refer to such a patient as treatment naïve.

The methods of the invention that concern diagnosing and/or treating acancer described herein (or use of a compound or compounds for suchpurposes) may specifically exclude any one or more of the types ofcancers described herein. For example, the invention features methods oftreating cancer by administering a compound of Formula (I), (Ia), aspecies thereof, or a specified form thereof, with the proviso that thecancer is not a breast cancer; with the proviso that the cancer is not abreast cancer or a leukemia; with the proviso that the cancer is not abreast cancer, a leukemia, or an ovarian cancer; and so forth, withexclusions selected from any of the cancers listed herein and with thesame notion of variable exclusion from lists of elements relevant toother aspects of the invention (e.g., chemical substituents of acompound described herein or components of kits and pharmaceuticalcompositions). Thus, where elements are presented as lists (e.g., inMarkush group format), every possible subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group.

In one aspect, the invention features the use of a compound of Formula(I), (Ia), a species thereof, or a specified form thereof in treatingcancer in a patient who has been identified by analyzing the biomarkerBCL2, RB1, RBL1, RBL2, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B,CDKN1C, or FBXW7 in a biological sample containing cancer cells or ctDNAfrom the patient. Analyzing the biomarker can include analyzing itssequence to detect a mutation or determining CNA, association with a SE,RNA expression level (e.g., mRNA expression) or another featuredescribed above as indicating the state of the biomarker. A patientidentified by analyzing BCL2, RB1, RBL1, RBL2, CDKN2A, CDKN2B, CDKN2C,CDKN2D, CDKN1A, CDKN1B, CDKN1C, or FBXW7 can be: treated with aplatinum-based therapeutic agent (e.g., carboplatin, cisplatin, oroxaliplatin) as a second agent; a patient whose cancer has developedresistance to a platinum-based therapeutic agent (e.g., carboplatin,cisplatin, or oxaliplatin); or a patient undergoing treatment with aCDK4/6 inhibitor used alone or in combination with one or more of anaromatase inhibitor, a selective estrogen receptor modulator (SERM),selective estrogen receptor degrader (SERD), or estrogen suppressant,any of which may be selected from the descriptions of such agentsprovided herein or known in the art. The patient's cancer may havebecome resistant to the CDK4/6 inhibitor or be at risk of becoming so.In the context of these uses (e.g., where the patient has beenidentified by analyzing the biomarker BCL2, RB1, RBL1, RBL2, CDKN2A,CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C, or FBXW7), the cancercan be a breast cancer (e.g., a triple negative breast cancer (TNBC),HR+, or other type of breast cancer described herein), an ovarian cancer(e.g., HGSOC), a lung cancer (e.g., SCLC, NSCLC or other lung cancerdescribed herein), retinoblastoma, or a blood cancer (e.g., acutemyeloid leukemia (AML)).

The methods of treating such a patient include a step of administeringan effective amount of a compound of Formula (I), (Ia), a speciesthereof or a specified form thereof, optionally within a pharma-ceuticalcomposition described herein and/or according to a dosing regimendescribed herein.

In another aspect, the invention features the use of a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof intreating cancer in a patient who has been identified by analyzing thebiomarker CCNE1, CCNE2, RB1, CDK6, CCND1, CCND2, CCND3, or CCKN2A in abiological sample containing cancer cells or ctDNA from the patient.Analyzing the biomarker can include analyzing its sequence to detect amutation or determining CNA, association with a SE, RNA expression level(e.g., mRNA expression) or another feature described above as indicatingthe state of the biomarker. A patient identified by analyzing CCNE1,CCNE2, RB1, CDK6, CCND1, CCND2, CCND3, or CCKN2A can be a patient whohas undergone, is presently undergoing, or who will undergo (e.g., hasbeen prescribed) treatment with a Bcl-2 inhibitor, such as venetoclax, aSERM, such as tamoxifen, a SERD, such as fulvestrant, or a PARPinhibitor, such as olaparib or niraparib. In the context of thesemethods, the patient may have a breast cancer (e.g., TNBC or an HR+breast cancer), lymphoma, melanoma (e.g., familial melanoma), ovariancancer (e.g., HGSOC), or pancreatic cancer (e.g., PDAC). For example,where the biomarker is CDKN2A, the patient may have TNBC, PDAC, orHGSOC. For example, where the biomarker is CCNE1, the patient may haveTNBC, HGSOC, melanoma (e.g., familial melanoma), or lymphoma. As notedabove, one of ordinary skill will recognize, as is well established inthe art, the relationship between a given gene and the protein itencodes. Thus, it will be clear that our reference to, for example, “thebiomarker BCL2” encompasses analysis of the biomarker gene BCL2-like 1and the biomarker protein (BCL2) encoded thereby; “the biomarker CCNE1”encompasses analysis of the biomarker gene CCNE1 and the biomarkerprotein (cyclin E1) encoded thereby; and so forth. The methods oftreating such a patient include a step of administering an effectiveamount of a compound of Formula (I), (Ia), a species thereof or aspecified form thereof, optionally within a pharmaceutical compositiondescribed herein and/or according to a dosing regimen described herein.

In another aspect, the invention features the use of a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof intreating cancer in a patient who has been identified by analyzing thebiomarker MYC (see Kalkat et al., Genes 8(6):151, 2017), CDK1, CDK2,CDK4, CDK17, CDK18, CDK19, CCNA1, CCNB1, ESR-1 or FGFR1 in a biologicalsample containing cancer cells or ctDNA from the patient. Analyzing thebiomarker can include analyzing any mutations within MYC, CDK1, CDK2,CDK4, CDK17, CDK18, CDK19, CCNA1, CCNB1, ESR-1 or FGFR1 or determiningCNA, association with a SE, RNA expression level (e.g., mRNA expression)or another feature described above as indicating the state of thebiomarker. The patient may have a breast cancer (e.g., TNBC or anovarian cancer (e.g., HGSOC) and may be resistant to a platinum-basedanti-cancer agent, such as carboplatin, cisplatin, or oxaliplatin,resistant to gemcitabine, resistant to a PARP inhibitor, such asolaparib or niraparib, or resistant to a taxane, such as paclitaxel. Themethods of treating such a patient include a step of administering aneffective amount of a compound of Formula (I), (Ia), a species thereofor a specified form thereof, optionally within a pharmaceuticalcomposition described herein and/or according to a dosing regimendescribed herein. C-myc encodes at least two phosphoproteins withapparent molecular weights of 62,000 and 66,000 (see Ramsay et al.,Proc. Natl. Acad. Sci. (USA) 81(24):7742-7746, 1984), and it has beendetermined through H3K27Ac ChIP-seq (ChIP-sequencing) methods that thereis a SE locus associated with the MYC gene at chr8:128628088-128778308(Gencode v19 annotation of the human genome build hg19/GRCh37).

In another aspect, the invention features the use of a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof intreating cancer in a patient who has been identified by analyzing thebiomarker CDK7 or CDK9. Analyzing the biomarker can include analyzingany mutations within CDK7 or CDK9 or determining CNA, association with aSE, RNA expression level (e.g., mRNA expression) or another featuredescribed above as indicating the state of the biomarker. Where thebiomarker is CDK7 or CDK9, the patient may have a lymphoma and thediagnosing/identifying step may more specifically be based on analysisof CDK7 (e.g., the level of CDK7 mRNA); the patient may have a breastcancer (e.g., TNBC), with the diagnosing/identifying step morespecifically based on CDK9 (e.g., the level of CDK9 mRNA); the patientmay have a TNBC or a lung cancer (e.g., SCLC), with the diagnosing stepmore specifically be based CDK19 (e.g., on the level of CDK19 mRNA). Themethods of treating such a patient include a step of administering aneffective amount of a compound of Formula (I), (Ia), a species thereofor a specified form thereof, optionally within a pharmaceuticalcomposition described herein and/or according to a dosing regimendescribed herein.

In another aspect, the invention features the use of a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof intreating cancer in a patient who has been identified by analyzing thebiomarker BRAF, E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, or E2F8 in abiological sample containing cancer cells or ctDNA from the patient.Analyzing the biomarker can include analyzing its sequence to detect amutation or determining CNA, association with a SE, RNA expression level(e.g., mRNA expression) or another feature described above as indicatingthe state of the biomarker. A patient identified by analyzing BRAF,E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, or E2F8 (by virtue of having afeature equal to or above a pre-determined threshold, as describedherein) can be a patient who has undergone, is presently undergoing, orwho will undergo (e.g., has been prescribed) treatment with a PI3Kinhibitor, such as alpelisib or capecitabine, a platinum-basedanti-cancer agent, such as carboplatin, cisplatin, or oxaliplatin, orvincristine. In the context of these methods, the patient may have amelanoma, lung cancer (e.g., NSCLC), GI tract cancer (e.g., CRC),thyroid cancer, retinoblastoma, or leukemia (e.g., hairy cell leukemia).The methods of treating such a patient include a step of administeringan effective amount of a compound of Formula (I), (Ia), a speciesthereof or a specified form thereof, optionally within a pharmaceuticalcomposition described herein and/or according to a dosing regimendescribed herein.

A compound or other composition described herein (e.g., a pharmaceuticalcomposition comprising a compound of Formula (I), (Ia), a speciesthereof or specified form thereof) can be administered in a combinationtherapy (e.g., as defined and further described herein) with a secondagent described herein or a plurality thereof (i.e., a patientidentified as described herein may be treated with first, second, andthird agents). The additional/second agent employed in a combinationtherapy is most likely to achieve a desired effect for the same disorder(e.g., the same cancer), however it may achieve different effects thataid the patient. Accordingly, the invention features pharmaceuticalcompositions containing a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof (e.g., a pharmaceuticallyacceptable salt), optionally in a therapeutically effect amount, for usein treating a patient identified as described herein. The pharmaceuticalcompositions may optionally include any of the additional/second agentsdescribed herein and will include a pharmaceutically acceptable carrier.The second/additional agent can be selected from a Bcl-2 inhibitor suchas venetoclax, a PARP inhibitor such as olaparib or niraparib, aplatinum-based anti-cancer agent such as carboplatin, cisplatin, oroxaliplatin, a taxane such as docetaxel or paclitaxel (or paclitaxelprotein-bound (available as Abraxane®)), a CDK4/6 inhibitor such aspalbociclib, ribociclib, abemaciclib, or trilaciclib, a selectiveestrogen receptor modulator (SERM) such as tamoxifen (available underthe brand names Nolvadex™ and Soltamox™), raloxifene (available underthe brand name Evista™), and toremifene (available as Fareston™) and aselective estrogen receptor degrader such as fulvestrant (available asFaslodex™), each in a therapeutically effective amount.

Unless otherwise specified, when employing a combination of a compoundof Formula (I), (Ia), a species thereof, or a specified variant thereofand a second therapeutic agent in a method of the invention, the secondtherapeutic agent can be administered concurrently with, prior to, orsubsequent to a compound of Formula (I), (Ia), a species thereof, or aspecified form thereof. The second therapeutic pharmaceutical agent maybe administered at a dose and/or on a time schedule determined for thatpharmaceutical agent. An additional/second therapeutic agent may also beadministered together with the compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof in a single dosage form oradministered separately in different dosage forms. In general, andwithout limitation, it is expected that the second therapeutic agentsutilized in combination with a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof will be utilized at levels that donot exceed the levels at which they are utilized individually. In someembodiments, the levels of the second therapeutic agent utilized incombination will be lower than those utilized in a monotherapy due tosynergistic effects.

In particular combination therapies for a patient identified asdescribed herein: (a) the cancer is TNBC, an ER+ breast cancer,pancreatic cancer (e.g., PDAC), or a squamous cell cancer of the head orneck and the second agent is a CDK4/6 inhibitor; (b) the cancer is abreast cancer (e.g., TNBC) or an ovarian cancer and the second agent isa PARP inhibitor; (c) the cancer is a leukemia (e.g., AML) and thesecond agent is a FLT3 inhibitor; (d) the cancer is an ovarian cancer(e.g., HGSOC) and the second agent is a platinum-based anti-canceragent; (e) the cancer is a breast cancer (e.g., TNBC), a leukemia (e.g.,AML), Ewing's sarcoma, or an osteosarcoma and the second agent is a BETinhibitor; (f) the cancer is a breast cancer (e.g., TNBC), a leukemia(e.g., AML), an ovarian cancer (e.g., HGSOC), or a lung cancer (e.g.,NSCLC) and the second agent is a Bcl-2 inhibitor. In particularembodiments, the cancer is AML and the second agent is a Bcl-2inhibitor, such as venetoclax; the cancer is an epithelial ovariancancer, a fallopian tube cancer, a primary peritoneal cancer, a triplenegative breast cancer or a Her2⁺/ER⁻/PR⁻ breast cancer and the secondagent is a PARP inhibitor, such as olaparib or niraparib; the cancer isan ovarian cancer and the second agent is a platinum-based anti-canceragent, such as carboplatin, cisplatin, or oxaliplatin. As noted above,and regardless of the biomarker analyzed or the type of cancer inquestion, a method of treatment can either be carried out on anidentified patient without an explicit step of analyzing the biomarkeror with an explicit step in which the biomarker is analyzed (e.g., byobtaining a biological sample from a patient).

With regard to combination therapies, a patient identified as describedherein can be treated with a combination of a compound of Formula (I),(Ia), a species thereof, or a specified form thereof and one or more ofa second agent that can be, but is not limited to, a Bcl-2 inhibitorsuch as APG-1252, APG-2575, BP1002 (prexigebersen), the antisenseoligonucleotide known as oblimersen (G3139), S55746/BCL201, orvenetoclax (e.g., venetoclax tablets marketed as Venclexta®); a CDK9inhibitor such as alvocidib/DSP-2033/flavopiridol, AT7519, AZD5576,BAY1251152, BAY1143572, CYC065, nanoflavopiridol, NVP2, seliciclib(CYC202), TG02, TP-1287, VS2-370 or voruciclib (formerly P1446A-05); ahormone receptor (e.g., estrogen receptor) degradation agent, such asfulvestrant (e.g., marketed as Faslodex® and others); a Flt3 (FMS-liketyrosine kinase 3) inhibitor such as CDX-301, CG′806, CT053PTSA,crenolanib (e.g., crenolanib besylate), ENMD-2076, FF-10101-01, FLYSYN,gilteritinib (ASP2215), HM43239, lestautinib, ponatinib (e.g., marketedas Iclusig®, previously AP24534), NMS-088, sorafenib (e.g., marketed asNexavar®), sunitinib, pacritinib, pexidartinib/PLX3397, quizartinib,midostaurin (e.g., marketed as Rydapt®), SEL24, SKI-G-801, or SKLB1028;a PARP inhibitor such as olaparib (e.g., marketed as Lynparza®),rucaparib (e.g., marketed as Rubraca®), talazoparib (e.g., marketed asTalzenna®), veliparib (ABT-888), or niraparib (e.g., marketed asZejula®); a BET inhibitor such as ABBV-075, BAY-299, BAY-1238097,BMS-986158, CPI-0610, CPI-203, FT-1101, GS-5829, GSK-2820151,GSK-525762, I-BET151, I-BET762, INCB054329, JQ1, MS436, OTX015, PFI-1,PLX51107, RVX2135, TEN-010, ZEN-3694, or a compound disclosed in U.S.application Ser. No. 12/810,564 (now U.S. Pat. No. 8,476,260), which ishereby incorporated herein by reference in its entirety; aplatinum-based therapeutic agent such as cisplatin, oxaliplatin (e.g.,marketed as Eloxatin®), nedaplatin, carboplatin (e.g., marketed asParaplatin®), phenanthriplatin, picoplatin, satraplatin (JM216), ortriplatin tetranitrate; a CDK4/6 inhibitor such as BPI-1178, G1T38,palbociclib (e.g., marketed as Ibrance®), ribociclib (e.g., marketed asKisqali®), ON 123300, trilaciclib, or abemaciclib (e.g., marketed asVerzenio®); a MEK inhibitor such as trametinib (e.g., marketed asMekinist®), cobimetinib (available as Cotellic®), or binimetinib(Braftovi®), useful in combination with a compound of Formula (I), (Ia),a species thereof, or specified form thereof, in treating, e.g.,melanoma); or a phosphoinositide 3-kinase (PI3 kinase) inhibitor,optionally of Class I (e.g., Class IA) and/or optionally directedagainst a specific PI3K isoform. The PI3K inhibitor can be apitolisib(GDC-0980), idelalisib (e.g., marketed as Zydelig®), copanlisib (e.g.,marketed as Aliqopa®), duvelisib (e.g., marketed as Copiktra®),pictilisib (GDC-0941), or alpelisib (e.g., marketed as Piqray®). Inother embodiments, the additional/second agent can be capecitabine(e.g., marketed as Xeloda®). Such PI3K inhibitors can be combined with acompound of Formula (I), (Ia), a species thereof or specified formthereof in treating, e.g., HR+ breast cancer, TNBC, lymphoma (e.g.,follicular lymphoma or non-Hodgkin lymphoma), or leukemia (e.g., CLL).In other embodiments, the additional/second agent can be gemcitabine(combined with a compound of the invention to treat, e.g., TNBC, CRC,SCLC, or a pancreatic cancer (e.g., PDAC)). In other embodiments, theadditional/second agent can be an antimetabolite, such as the pyrimidineanalog 5-fluorouracil (5-FU), which may be used in combination with acompound of Formula (I), (Ia), a species thereof, or a specified formthereof, and one or more of leucovorin, methotrexate, or oxaliplatin. Inother embodiments, the additional/second agent can be an aromataseinhibitor, such as exemestane or anastrasole. In other embodiments, theadditional/second agent is an inhibitor of the PI3K/AKT/mTOR pathway(e.g., gedatolisib). In one embodiment, the methods encompass the use ofor administration of a compound of Formula (I), (Ia), a species thereofor a specified form thereof, to a patient identified as describedherein, in combination with a MEK inhibitor, such as trametinib(available as Mekinist®), cobimetinib (available as Cotellic®), orbinimetinib (available as Braftovi®)

APG-1252 is a dual Bcl-2/Bcl-xL inhibitor that has shown promise inearly clinical trials when patients having SCLC or another solid tumorwere dosed between 10-400 mg (e.g., 160 mg) intravenously twice weeklyfor three weeks in a 28-day cycle (see Lakhani et al., J. Clin. Oncol.36:15_suppl, 2594, and ClinicalTrials.gov identifier NCT03080311).APG-2575 is a Bcl-2 selective inhibitor that has shown promise inpreclinical studies of FL and DLBCL in combination with ibrutinib (seeFang et al., AACR Annual Meeting 2019, Cancer Res. 79(13 Suppl):AbstractNo. 2058) and has begun clinical trials as a single-agent treatment forpatients with blood cancers; in a dose escalation study, patients aregiven 20 mg, once daily, by mouth, for four consecutive weeks as onecycle. Escalations to 50, 100, 200, 400, 600 and 800 mg are planned toidentify the MTD (see ClinicalTrials.gov identifier NCT03537482). BP1002is an uncharged P-ethoxy antisense oligodeoxynucleotide targeted againstBcl-2 mRNA that may have fewer adverse effects than other antisenseanalogs and has shown promise in inhibiting the growth of human lymphomacell lines inclubated with BP1002 for four days and of CJ cells(transformed FL cells) implanted into SCID mice (see Ashizawa et al.,AACR Annual Meeting 2017, Cancer Res. 77(13 Suppl):Abstract No. 5091).BP1002 has also been administered in combination with cytarabine (LDAC)to patients having AML (see ClinicalTrials.gov identifier NCT04072458).S55746/BCL201 is an orally available, selective Bcl-2 inhibitor that, inmice, demonstrated anti-tumor efficacy in two blood cancer xenograftmodels (Casara et al., Oncotarget 9(28):20075-88, 2018). A phase Idose-escalation study was designed to administer film-coated tabletscontaining 50 or 100 mg of S55746, in doses up to 1500 mg, to patientswith CLL or a B cell NHL including FL, MCL, DLBCL, SLL, MZL, and MM (seeClinicalTrials.gov identifier NCT02920697). Venetoclax tablets have beenapproved for treating adult patients with CLL or SLL and, in combinationwith azacytidine, or decitabine, or low-dose cytarabine, for treatingnewly-diagnosed AML in patients who are at least 75 years old or whohave comorbidities that preclude the use of intensive inductionchemotherapy. Dosing for CLL/SLL can follow the five-week ramp-upschedule and dosing for AML can follow the four-day ramp-up, bothdescribed in the product insert, together with other pertinentinformation (see also U.S. Pat. Nos. 8,546,399; 9,174,982; and9,539,251, which are hereby incorporated by reference in theirentireties). Alvocidib was studied in combination withcytarabine/mitoxantrone or cytarabine/daunorubicin in patients with AML,with the details of administration being available at ClinicalTrials.govwith the identifier NCT03563560 (see also Yeh et al., Oncotarget6(5):2667-2679, 2015, Morales et al., Cell Cycle 15(4):519-527, 2016,and Zeidner et al., Haematologica 100(9):1172-1179, 2015). AT7519 hasbeen administered in a dose escalation format to eligible patientshaving refractory solid tumors. While there was some evidence ofclinical activity, the appearance of QTc prolongation precluded furtherdevelopment at the dose schedule described by Mahadevan et al. (J. Clin.Oncol. ASCO Abstract No. 3533; see also Santo et al., Oncogene29:2325-2336, 2010, describing the preclinical activity of AT7519 inMM). AZD5576 induced apoptosis in breast and lung cancer cell lines atthe nanomolar level (see Li et al., Bioorg. Med. Chem. Lett.27(15):3231-3237, 2017) and has been examined alone and in combinationwith acalabrutinib for the treatment of NHL (see AACR 2017 Abstract No.4295). BAY1251152 was the subject of a phase I clinical trial tocharacterize the MTD in patients with advanced blood cancers; the agentwas infused weekly in 21-day cycles (see ClinicalTrials.gov identifierNCT02745743; see also Luecking et al., AACR 2017 Abstract No. 984).Voruciclib is a clinical stage oral CDK9 inhibitor that represses MCL-1and sensitizes high-risk DLBCL to BCL2 inhibition. Dey et al.(Scientific Reports 7:18007, 2017) suggest that the combination ofvoruciclib and venetoclax is promising for a subset of high-risk DLBCLpatients (see also ClinicalTrials.gov identifier NCT03547115).Fulvestrant has been approved for administration to postmenopausal womenwith advanced hormone receptor (HR)-positive, HER2-negative breastcancer, with HR-positive metastatic breast cancer whose diseaseprogressed after treatment with other anti-estrogen therapies, and incombination with palbociclib (Ibrance®). Fulvestrant is administered byintramuscular injection at 500 or 250 mg (the lower dose beingrecommended for patients with moderate hepatic impairment) on days 1,15, and 29, and once monthly thereafter (see the product insert foradditional information; see also U.S. Pat. Nos. 6,744,122; 7,456,160;8,329,680; and 8,466,139, each of which are hereby incorporated byreference herein in their entireties). Ponatinib has been administeredin clinical trials to patients with CML or ALL (see ClinicalTrials.govidentifiers NCT0066092072, NCT012074401973, NCT02467270, NCT03709017,NCT02448095, NCT03678454, and NCT02398825) as well as solid tumors, suchas biliary cancer and NSCLC (NCT02265341, NCT02272998, NCT01813734,NCT02265341, NCT02272998, NCT01813734, NCT02265341, NCT02272998,NCT01813734, NCT01935336, NCT03171389, and NCT03704688; see also thereview article by Tan et al., Onco. Targets Ther. 12:635-645, 2019).Additional information regarding the dosing regimen can be found in theproduct insert; see also U.S. Pat. Nos. 8,114,874; 9,029,533; and9,493,470, each of which is hereby incorporated by reference herein inits entirety. Sorafenib has been approved for the treatment of kidneyand liver cancers, AML, and radioactive iodine resistant advancedthyroid cancer, and a clinical trial was initiated in patients withdesmoid-type fibromatosis (see ClinicalTrials.gov identifierNCT02066181). Information regarding dosage can be found in the productinsert, which advises administration of two, 400 mg tablets twice daily;see also U.S. Pat. Nos. 7,235,576; 7,351,834; 7,897,623; 8,124,630;8,618,141; 8,841,330; 8,877,933; and 9,737,488, each of which is herebyincorporated by reference herein in its entirety. Midostaurin has beenadministered to patients having AML, MDS, or systemic mastocytosis, andhas been found to significantly prolong survival of FLT3-mutated AMLpatients when combined with conventional induction and consolidationtherapies (see Stone et al., ASH 57th Annual Meeting, 2015 and Galloglyet al., Ther. Adv. Hematol. 8(9):245-251, 2017; din see also the productinsert, ClinicalTrials.gov identifier NCT03512197, and U.S. Pat. Nos.7,973,031; 8,222,244; and 8,575,146, each of which is herebyincorporated by reference herein in its entirety. Alpelisib is a kinaseinhibitor indicated in combination with fulvestrant for the treatment ofpostmenopausal women, and men, with HR+/HER2−/PIK3CA-mutated, advancedor metastatic breast cancer as deteted by an FDA-approved test followingprogression on or after an endocrine-based regimen. The recommended doseis 300 mg (two 150 mg tablets) taken orally once daily with food, which,as for all chemotherapeutic agents, may be interrupted, reduced, ordiscontinued to manage adverse reactions. Paclitaxel is supplied as anonaqueous solution intended for dilution with a suitable parenteralfluid prior to intravenous infusion. Under the brand name Taxol®, it issupplied in 30 mg, 100 mg, and 300 mg vials and can be used in acombination therapy described herein to treat a variety of cancers,including those of the bladder, breast, esophagus, fallopian tube orovary, lung, skin (melanoma), and prostate. Palbociclib has beenapproved for use in HR+/HER2− advanced or metastatic breast cancer at arecommended dose of 125 mg daily, by mouth. It can be used to treat apatient as identified herein with a compound of Formula (I), (Ia), aspecies thereof, or a specified form thereof, either alone or incombination with an aromatase inhibitor or fulvestrant. The informationprovided here and publicly available can be used to practice the methodsand uses of the invention. In case of doubt, the invention encompassescombination therapies that require a compound of the invention or aspecified form thereof and any one or more additional/second agents,which may be administered at or below a dosage currently approved forsingle use (e.g., as described above), to a patient as described herein.Triplet combinations include a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof with: alpelisib and fulvestrant oralpelisib and a taxane (for, e.g., treating NSCLC).

Where the combination therapy employs a compound of the invention and: aCDK4/6 inhibitor, the patient can have a breast cancer (e.g., TNBC or anER+ breast cancer), pancreatic cancer, lung cancer (e.g., SCLC orNSCLC), or squamous cell cancer of the head and neck; a CDK9 inhibitor,the patient can have a breast cancer and, more specifically, aHer2⁺/ER⁻/PR⁻ breast cancer; a Flt3 inhibitor (e.g., midostaurin), thepatient can have a hematological cancer (e.g., AML); a BET inhibitor,the patient can have a hematological cancer (e.g., AML), a breast cancer(e.g., TNBC), an osteosarcoma or Ewing's Sarcoma; a Bcl-2 inhibitor(e.g., venetoclax), the patient can have a breast cancer (e.g., TNBC),an ovarian cancer, a lung cancer (e.g., NSCLC) or a hematological cancer(e.g., AML); or a PARP inhibitor (e.g., niraparib or olaparib), thepatient can have a breast cancer (e.g., TNBC or Her2⁺/ER⁻/PR⁻ breastcancer), an ovarian cancer (e.g., an epithelial ovarian cancer), afallopian tube cancer, or a primary peritoneal cancer.

In some aspects relating to using RB-E2F pathway genes (or the proteinsthey encode) as biomarkers, the invention provides a method of treatinga patient having a cancer and identified as described herein, whichcomprises administering to a patient identified as having either (a) alevel of CCNE1 mRNA or protein in the cancer equal to or above apre-determined threshold; and/or (b) a level of RB1 mRNA or protein inthe cancer equal to or below a pre-determined threshold, an effectiveamount of a CDK7 inhibitor of Formula (I). In some aspects of theseembodiments, the method further comprises determining a level of RB1and/or CCNE1 mRNA or protein present in a sample of cancer cells fromthe patient. In various embodiments, the human patient is diagnosed ashaving a cancer sensitive to a CDK7 inhibitor responsive to thedetermination; is suffering from ovarian cancer; or is suffering from abreast cancer (e.g., TNBC or an HR⁺ breast cancer). In some embodiments,a compound of Formula (I), (Ia), a species thereof or a specified formthereof is co-administered with a PARP inhibitor. In some embodiments,the compound of Formula (I), (Ia), a species thereof or a specified formthereof is co-administered with a SERM (e.g., tamoxifen, raloxifene, ortoremifene), a SERD such fulvestrant, or an agent that inhibits theproduction of estrogen (e.g., an aromatase inhibitor such as anastrozole(available as Arimidex®), exemestane (available as Aromasin®), andletrozole (available as Femara®), optionally to treat a cancer that isrefractory to palbociclib. Our data indicate that a compound of Formula(I), (Ia), a species thereof, or a specified form thereof (e.g.,Compound 101) can induce deep and sustained TGI in combination withfulvestrant in palbociclib-resistant (PBR) ER+ breast cancer PDX models.Further, based on data with Compound 101, we believe compounds of theinvention can resensitize palbociclib- and fulvestrant-resistant(PBR/FSR) ER+ breast cancer PDX tumors to fulvestrant treatment. Inother embodiments, the invention provides methods of treating cancer ina patient identified as described herein by administering to the patienta combination of a compound of Formula (I), (Ia), a species thereof, ora specified form thereof and a platinum-based standard of care (SOC)anti-cancer agent for such cancer or a taxane. The cancer can be anovarian cancer and the SOC anti-cancer agent can be a platinum-basedanti-cancer agent (e.g., carboplatin, cisplatin, or oxaliplatin). Insome embodiments, the human patient is, has been determined to be, orhas become resistant (after some initial responsiveness) to theplatinum-based anti-cancer agent when administered as either amonotherapy or in combination with an anti-cancer agent other than aCDK7 inhibitor. In some aspects of this embodiment, the human patient isdetermined to have become resistant to the platinum-based anti-canceragent when administered as a monotherapy or in combination with ananti-cancer agent other than a CDK7 inhibitor after some initialefficacy of that prior treatment. In some aspects of this embodiment,the SOC anti-cancer agent is a taxane (e.g., paclitaxel).

The invention also provides methods of treating a biomarker-identifiedHR⁺ breast cancer in a human patient selected on the basis of beingresistant to treatment with a CDK4/6 inhibitor comprising the step ofadministering to the patient a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof. In some embodiments, prior toadministration of the compound of Formula (I), (Ia), a species thereof,or a specified form thereof, the patient is, has been determined to be,or has become resistant (after some initial responsiveness) to a priortreatment with a CDK4/6 inhibitor alone or in combination with anotherSOC agent for breast cancer other than a CDK7 inhibitor, such as anaromatase inhibitor (e.g., letrozole, anastrozole) or a SERM (e.g.,tamoxifen, raloxifene, or toremifene), SERD (e.g., fulvestrant), orestrogen suppressant such as anastrozole (available as Arimidex®),exemestane (available as Aromasin®), or letrozole (available asFemara®). In other words, the identified patient is selected fortreatment with a compound of Formula (I), (Ia), a species thereof, or aspecified form thereof on the basis of being resistant to priortreatment with a CDK4/6 inhibitor alone or in combination with anotherSOC agent for breast cancer other than a CDK7 inhibitor. In someembodiments, the compound of Formula (I), (Ia), a species thereof, or aspecified form thereof is co-administered with another SOC agent, suchas an aromatase inhibitor (e.g. anastrozole, exemestane, or letrozole)and/or a SERM or SERD, e.g., as described herein, or a second linetreatment after failure on an aromatase inhibitor or fulvestrant. Insome embodiments, prior to administration of the compound of Formula(I), (Ia), a species thereof, or a specified form thereof, the patientis, has been determined to be, or has become resistant (after someinitial responsiveness) to treatment with a CDK4/6 inhibitor alone or incombination with another SOC agent for breast cancer other than a CDK7inhibitor, such as an aromatase inhibitor (e.g., anastrozole,exemestane, or letrozole), or a SERM or SERD such as tamoxifen orfulvestrant; and the compound of Formula (I), (Ia), a species thereof,or a specified form thereof is co-administered with a SOC agent forbreast cancer (e.g., a second line treatment after failure of anaromatase inhibitor or a SERM or SERD such as tamoxifen or fulvestrant.

An enhancer or SE can be identified by various methods known in the art(see Hinsz et al., Cell, 155:934-947, 2013; McKeown et al., CancerDiscov., 7(10):1136-53, 2017; and PCT/US2013/066957, each of which arehereby incorporated herein by reference in their entireties).Identifying a SE can be achieved by obtaining a biological sample from apatient (e.g., from a biopsy or other source, as described herein). Theimportant metrics for enhancer measurement occur in two dimensions:along the length of the DNA over which genomic markers (e.g., H3K27Ac)are contiguously detected and the compiled incidence of genomic markerat each base pair along that span of DNA, the compiled incidenceconstituting the magnitude. The measurement of the area under the curve(“AUC”) resulting from integration of length and magnitude analysesdetermines the strength of the enhancer. The strength of the BRAF, MYC,CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3,CCNE1, or CCNE2) SEs relative to an appropriate reference can be used todiagnose (stratify) a patient and thereby determine whether a patient islikely to respond well to a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof. It will be readily apparent to oneof ordinary skill in the art, particularly in view of the instantspecification, that if the length of DNA over which the genomic markersis detected is the same for each of BRAF, MYC, CDK1, CDK2, CDK4, CDK6,CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, orcertain genes encoding an E2F pathway member (E2F1, E2F2, E2F3, E2F4,E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3, CCNE1, or CCNE2) and thereference/control, then the ratio of the magnitude of the BRAF, MYC,CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3,CCNE1, or CCNE2) SE relative to the control will be equivalent to thestrength and may also be used to determine whether a patient will beresponsive to a compound of Formula (I), (Ia), a species thereof, or aspecified form thereof. The strength of the BRAF, MYC, CDK1, CDK2, CDK4,CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,PIK3CA, or certain genes encoding an E2F pathway member (E2F1, E2F2,E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2, CCND3, CCNE1, orCCNE2) SE in a cell can be normalized before comparing it to othersamples. Normalization is achieved by comparison to a region in the samecell known to comprise a ubiquitous SE or enhancer that is present atsimilar levels in all cells. One example of such a ubiquitoussuper-enhancer region is the MALAT1 super-enhancer locus(chr11:65263724-65266724) (genome build hg19).

It has been determined through H3K27Ac ChIP-seq (ChIP-sequencing)methods that there is a SE locus associated with the CDK18 gene atchr1:205399084-205515396; a SE locus associated with the CDK19 gene atchr6:110803523-110896277; a SE locus associated with the CCNE1 gene atchr19:30418503-30441450; and a SE locus associated with the FGFR1 geneat chr8:38233326-38595483. All loci are based on the Gencode v19annotation of the human genome build hg19/GRCh37.

ChIP-seq is used to analyze protein interactions with DNA by combiningchromatin immunoprecipitation (ChIP) with massively parallel DNAsequencing to identify the binding sites of DNA-associated proteins. Itcan be used to map global binding sites precisely for any protein ofinterest. Previously, ChIP-on-chip was the most common techniqueutilized to study these protein-DNA relations. Successful ChIP-seq isdependent on many factors including sonication strength and method,buffer compositions, antibody quality, and cell number (see, e.g.,Furey, Nature Reviews Genetics 13:840-852, 2012); Metzker, NatureReviews Genetics 11:31-46, 2010; and Park, Nature Reviews Genetics10:669-680, 2009). Genomic markers other than H3K27Ac that can be usedto identify SEs using ChIP-seq include P300, CBP, BRD2, BRD3, BRD4,components of the mediator complex (Loven et al., Cell, 153(2):320-334,2013), histone 3 lysine 4 monomethylated (H3K4me1), and othertissue-specific enhancer tied transcription factors (Smith andShilatifard, Nature Struct. Mol. Biol., 21(3):210-219, 2014; and Pottand Lieb, Nature Genetics, 47(1):8-12, 2015). Quantification of enhancerstrength and identification of SEs can be determined using SE scores(McKeown et al., Cancer Discov. 7(10):1136-1153, 2017; DOI:10.1158/2159-8290.CD-17-0399).

In some instances, H3K27Ac or other marker ChIP-seq data SE maps of theentire genome of a cell line or a patient sample already exist. Onewould then simply determine whether the strength or ordinal rank of theenhancer or SE in such maps at the chr8:128628088-128778308 (genomebuild hg19) locus was equal to or above the pre-determined thresholdlevel. In some embodiments, one would simply determine whether thestrength, or ordinal rank of the enhancer or super-enhancer in such mapsat the chr1:205399084-205515396 (genome build hg19) locus was equal toor above the pre-determined threshold level.

It should be understood that the specific chromosomal location of BRAF,MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1,CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathwaymember (E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CCND1, CCND2,CCND3, CCNE1, or CCNE2) and MALAT1 may differ for different genomebuilds and/or for different cell types. The same is true for BCL2-like1, CDK7, CDK9, CDKN2A, and RB1 or another E2F pathway member that isunderexpressed in cancer (E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7,E2F8, CCND1, CCND2, CCND3, CCNE1, or CCNE2). However, one skilled in theart, particularly in view of the instant specification, can determinesuch different locations by locating in such other genome buildsspecific sequences corresponding to the loci in genome build hg 19.

Other methods that can be used to identify SEs in the context of thepresent methods include chromatin immunoprecipitation (Delmore et al.,Cell, 146(6):904-917, 2011), chip array (ChIP-chip), and chromatinimmunoprecipitation followed by qPCR (ChIP-qPCR) using the sameimmunoprecipitated genomic markers and oligonucleotide sequences thathybridize to the chr8:128628088-128778308 (genome build hg19) MYC locusor chr1:205399084-205515396 (genome build hg19) CDK18 locus (forexample). In the case of ChIP-chip, the signal is typically detected byintensity fluorescence resulting from hybridization of a probe and inputassay sample as with other array-based technologies. For ChIP-qPCR, adye that becomes fluorescent after intercalating the double stranded DNAgenerated in the PCR reaction is used to measure amplification of thetemplate.

In some embodiments, determination of whether a cell has a BRAF, MYC,CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(see above) SE strength equal to or above a requisite threshold level isachieved by comparing BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17,CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certaingenes encoding an E2F pathway member (see above) enhancer strength in atest cell to the corresponding BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7,CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, orcertain genes encoding an E2F pathway member (see above) strength in apopulation of cell samples, wherein each of the cell samples is obtainedfrom a different source (e.g., a different patient, a different cellline, a different xenograft) reflecting the same disease to be treated.In some embodiments, only primary tumor cell samples from patients areused to determine the threshold level. In some aspects of theseembodiments, at least some of the samples in the population will havebeen tested for responsiveness to a specific CDK7 inhibitor (e.g., acompound of Formula (I), (Ia), a species thereof or a specified formthereof) to establish: (a) the lowest M BRAF, MYC, CDK1, CDK2, CDK4,CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,PIK3CA, or certain genes encoding an E2F pathway member (see above)enhancer strength of a sample in the population that responds to thatspecific compound (“lowest responder”); and, optionally, (b) the highestBRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1,CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2Fpathway member (see above) enhancer strength of a sample in thepopulation that does not respond to that specific compound (“highestnon-responder”). In these embodiments, a cutoff of BRAF, MYC, CDK1,CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1,FGFR1, PIK3CA, or certain genes encoding an E2F pathway member (seeabove) enhancer strength above which a test cell would be consideredresponsive to that specific compound is set: i) equal to or up to 5%above the BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19,CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding anE2F pathway member (see above) enhancer strength in the lowest responderin the population; or ii) equal to or up to 5% above the BRAF, MYC,CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(see above) enhancer strength in the highest non-responder in thepopulation; or iii) a value in between the BRAF, MYC, CDK1, CDK2, CDK4,CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,PIK3CA, or certain genes encoding an E2F pathway member (see above)enhancer strength of the lowest responder and the highest non-responderin the population.

In the above embodiments, not all of the samples in a populationnecessarily are to be tested for responsiveness to a specific CDK7inhibitor (e.g., a compound of Formula (I), (Ia), a species thereof or aspecified form thereof), but all samples are measured for BRAF, MYC,CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1,FGFR1, PICKCA, or certain genes encoding an E2F pathway member (seeabove) enhancer strength. In some embodiments, the samples are rankordered based on M BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18,CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genesencoding an E2F pathway member (see above) enhancer strength. The choiceof which of the three methods set forth above to use to establish thecutoff will depend upon the difference in BRAF, MYC, CDK1, CDK2, CDK4,CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, orcertain genes encoding an E2F pathway member (see above) enhancerstrength between the lowest responder and the highest non-responder inthe population and whether the goal is to minimize the number of falsepositives or to minimize the chance of missing a potentially responsivesample or patient. When the difference between the lowest responder andhighest non-responder is large (e.g., when there are many samples nottested for responsiveness that fall between the lowest responder and thehighest non-responder in a rank ordering of BRAF, MYC, CDK1, CDK2, CDK4,CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, orcertain genes encoding an E2F pathway member (see above) enhancerstrength), the cutoff is typically set equal to or is up to 5% above theBRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1,CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathwaymember (see above) enhancer strength in the lowest responder in thepopulation. This cutoff maximizes the number of potential responders.When this difference is small (e.g., when there are few or no samplesuntested for responsiveness that fall between the lowest responder andthe highest non-responder in a rank ordering of BRAF, MYC, CDK1, CDK2,CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,PIK3CA, or certain genes encoding an E2F pathway member (see above)enhancer strength), the cutoff is typically set to a value in betweenthe BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1,CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2Fpathway member (see above) enhancer strength of the lowest responder andthe highest non-responder. This cutoff minimizes the number of falsepositives. When the highest non-responder has a BRAF, MYC, CDK1, CDK2,CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1, ESR-1, FGFR1,PIK3CA, or certain genes encoding an E2F pathway member (see the Tableherein) enhancer strength that is greater than the lowest responder, thecutoff is typically set to a value equal to or up to 5% above the BRAF,MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(see above) enhancer strength in the highest non-responder in thepopulation. This method also minimizes the number of false positives.

In some embodiments, the methods discussed above can be employed tosimply determine if a diseased cell (e.g., a cancer cell) from a patienthas a SE associated with a biomarker gene described herein (e.g., BRAF,MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(see above) or a protein encoded thereby). The presence of the SEindicates that the patient is likely to respond well to a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof. Thecell is determined to have a SE associated with the biomarker (e.g.,BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1, CCNB1,CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathwaymember (see above) or a protein encoded thereby) when the enhancer has astrength that is equal to or above the enhancer associated with MALAT-1.In alternate embodiments, the cell is determined to have a SE associatedwith BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK17, CDK18, CDK19, CCNA1,CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2Fpathway member (see above) when the BRAF−, MYC−, CDK1−, CDK2−, CDK4−,CDK6−, CDK17−, CDK18−, CDK19−, CCNA1−, CCNB1−, CCNE1−, ESR−1−, FGFR1−,PIK3CA−, or certain genes encoding an E2F pathway member- (see above)associated enhancer has a strength that is at least 10-fold greater thanthe median strength of all of the enhancers in the cell. In otherembodiments, the cell is determined to have a SE associated with anaforementioned gene when the gene-associated enhancer has a strengththat is above the point where the slope of the tangent is 1 in arank-ordered graph of strength of each of the enhancers in the cell.

For any biomarker (e.g., in embodiments involving CDK18), the cutoffvalue for enhancer strength can be converted to a prevalence cutoff,which can then be applied to biomarker RNA levels (e.g., CDK18 mRNA)levels to determine a mRNA cutoff value in a given mRNA assay.

In some embodiments, biomarker mRNA levels in a patient (as assessed,e.g., in a biological sample obtained from the patient) are compared,using the same assay, to the same gene of interest/biomarker mRNA levelsin a population of patients having the same disease or condition toidentify likely responders to a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof. Analogous comparisons can be madewhen another feature of the biomarker is selected for analysis (e.g.,its copy number, chromosomal location, primary RNA level, or expressedprotein level). In embodiments where a biomarker (e.g., CDK18, CDK19,and CCNE1) correlates with (e.g., is one whose mRNA expressioncorrelates with) responsiveness to a compound of the invention, at leastsome of the samples in the population will have been tested forresponsiveness to the inhibitor (e.g., a compound of Formula (I), (Ia),a species thereof, or specified form thereof) to establish: (a) thelowest level (e.g., mRNA level) in a sample in the population thatresponds to that specific compound (“lowest mRNA responder”); and,optionally, (b) the highest level (e.g., highest mRNA level) in a samplein the population that does not respond to that specific compound(“highest mRNA non-responder”). In these embodiments, a cutoff ofbiomarker mRNA level above which a test cell would be consideredresponsive to that specific compound is set: i) equal to or up to 5%above the level (e.g., the mRNA level) in the lowest mRNA responder inthe population; or ii) equal to or up to 5% above the level (e.g., themRNA level) in the highest mRNA non-responder in the population; or iii)a value in between the level (e.g., mRNA level) of the lowest responder(e.g., lowest mRNA responder) and the highest responder (e.g., highestmRNA) non-responder in the population.

In embodiments where mRNA levels positively correlate with sensitivityto a compound of Formula (I), (Ia), a species thereof or a specifiedform thereof, not all of the samples in a population need to be testedfor responsiveness to the compound, but all samples are measured for thegene of interest mRNA levels. In some embodiments, the samples are rankordered based on gene of interest mRNA levels. The choice of which ofthe three methods set forth above to use to establish the cutoff willdepend upon the difference in gene of interest mRNA levels between thelowest mRNA responder and the highest mRNA non-responder in thepopulation and whether the cutoff is designed to minimize falsepositives or maximize the potential number of responders. When thisdifference is large (e.g., when there are many samples not tested forresponsiveness that fall between the lowest mRNA responder and thehighest mRNA non-responder in a rank ordering of mRNA levels), thecutoff is typically set equal to or up to 5% above the mRNA level in thelowest mRNA responder. When this difference is small (e.g., when thereare few or no samples untested for responsiveness that fall between thelowest mRNA responder and the highest mRNA non-responder in a rankordering of mRNA levels), the cutoff is typically set to a value inbetween the mRNA levels of the lowest mRNA responder and the highestmRNA non-responder. When the highest mRNA non-responder has a mRNA levelthat is greater than the lowest mRNA responder, the cutoff is typicallyset to a value equal to or up to 5% above the mRNA levels in the highestmRNA non-responder in the population.

In embodiments where a biomarker is one whose mRNA expression inverselycorrelates with responsiveness to a compound of Formula (I), (Ia), aspecies thereof, or a specified form thereof (i.e., BCL-xL, CDK7, CDK9,or an RB1 family member), at least some of the samples in the populationwill have been tested for responsiveness to the compound in order toestablish: (a) the highest mRNA level of a sample in the population thatresponds to that specific compound (“highest mRNA responder”); and,optionally, (b) the lowest mRNA level of a sample in the population thatdoes not respond to that specific compound (“lowest mRNAnon-responder”). In these embodiments, a cutoff of mRNA level abovewhich a test cell would be considered responsive to that specificcompound is set: i) equal to or up to 5% below the mRNA level in thehighest mRNA responder in the population; or ii) equal to or up to 5%below the mRNA level in the lowest mRNA non-responder in the population;or iii) a value in between the mRNA level of the lowest mRNAnon-responder and the highest mRNA responder and in the population.

In embodiments where mRNA levels inversely correlate with sensitivity toa compound of the invention, not all of the samples in a population needto be tested for responsiveness to the compound, but all samples aremeasured for the gene of interest mRNA levels. In some embodiments, thesamples are rank ordered based on gene of interest mRNA levels. Thechoice of which of the three methods set forth above to use to establishthe cutoff will depend upon the difference in gene of interest mRNAlevels between the highest mRNA responder and the lowest mRNAnon-responder in the population and whether the cutoff is designed tominimize false positives or maximize the potential number of responders.When this difference is large (e.g., when there are many samples nottested for responsiveness that fall between the highest mRNA responderand the lowest mRNA non-responder in a rank ordering of mRNA levels),the cutoff is typically set equal to or up to 5% below the mRNA level inthe highest mRNA responder. When this difference is small (e.g., whenthere are few or no samples untested for responsiveness that fallbetween the highest mRNA responder and the lowest mRNA non-responder ina rank ordering of mRNA levels), the cutoff is typically set to a valuein between the mRNA levels of the highest mRNA responder and the lowestmRNA non-responder. When the highest mRNA responder has a mRNA levelthat is lower than the lowest mRNA responder, the cutoff is typicallyset to a value equal to or up to 5% below the mRNA levels in the lowestmRNA non-responder in the population.

In embodiments involving CDK18, the cutoff for CDK18 mRNA levels may bedetermined using the prevalence cutoff established based on CDK18enhancer strength, as described above. In some embodiments, a populationis measured for mRNA levels and the prior determined prevalence cutoffis applied to that population to determine an mRNA cutoff level. In someaspects of these embodiments a rank-order standard curve of CDK18 mRNAlevels in a population is created, and the pre-determined prevalencecutoff is applied to that standard curve to determine the CDK18 mRNAcutoff level.

In some aspects of embodiments where a test cell or sample is comparedto a population, the cutoff mRNA level value(s) obtained for thepopulation is converted to a prevalence rank and the mRNA level cutoffis expressed as a percent of the population having the cutoff value orhigher, e.g., a prevalence cutoff. Without being bound by theory,applicants believe that the prevalence rank of a test sample and theprevalence cutoff in a population will be similar regardless of themethodology used to determine mRNA levels.

A patient can be identified as likely to respond well to a compound ofFormula (I), (Ia), a species thereof, or a specified form thereof if thestate of BRAF, MYC, CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19,CCNA1, CCNB1, CCNE1, ESR-1, FGFR1, PIK3CA, or certain genes encoding anE2F pathway member (see above) as determined by, e.g., RNA (e.g., mRNAlevels) in a biological sample from the patient) corresponds to (e.g.,is equal to or greater than) a prevalence rank in a population of about80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%,66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%,42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%,38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%,24%, 23%, 22%, 21%, or 20% as determined by the state of BRAF, MYC,CDK1, CDK2, CDK4, CDK6, CDK7, CDK17, CDK18, CDK19, CCNA1, CCNB1, CCNE1,ESR-1, FGFR1, PIK3CA, or certain genes encoding an E2F pathway member(see above), respectively, determined by assessing the same parameter(e.g., mRNA level(s)) in the population. A patient can be identified aslikely to respond well to a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof if the state of BCL2-like 1, CDK7,CDK9, CDKN2A, and RB (as determined by, e.g., RNA (e.g., mRNA) levels orcorresponding protein levels in a biological sample from the patient) isbelow a prevalence rank in a population of about 80%, 79%, 78%, 77%,76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%,48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%,34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or20% as determined by the state of BCL2-like 1, CDK7, CDK9, CDKN2A, andRB, respectively, determined by assessing the same parameter (e.g., mRNAlevel(s)) in the population. In some embodiments, the cutoff value orthreshold is established based on the biomarker (e.g., mRNA) prevalencevalue.

In still other embodiments, a population may be divided into threegroups: responders, partial responders and non-responders, and twocutoff values (or thresholds) or prevalence cutoffs (or thresholds) areset or determined. The partial responder group may include respondersand non-responders as well as those patients whose response to acompound of Formula (I), (Ia), a species thereof, or a specified formthereof was not as high as the responder group. This type ofstratification may be particularly useful when, in a population, thehighest mRNA non-responder has an mRNA level that is greater than thatof the lowest mRNA responder. In this scenario, for CDK18 or CDK19, thecutoff level or prevalence cutoff between responders and partialresponders is set equal to or up to 5% above the CDK18 or CDK19 mRNAlevel of the highest CDK18 or CDK19 mRNA non-responder; and the cutofflevel or prevalence cutoff between partial responders and non-respondersis set equal to or up to 5% below the CDK18 or CDK19 mRNA level of thelowest CDK18 or CDK19 mRNA responder. For BCL-xL, CDK7 or CDK9, thistype of stratification may be useful when the highest mRNA responder hasa mRNA level that is lower than that of the lowest mRNA non-responder.In this scenario, for BCL-xL, CDK7 or CDK9, the cutoff level orprevalence cutoff between responders and partial responders is set equalto or up to 5% below the mRNA level of the lowest mRNA non-responder;and the cutoff level or prevalence cutoff between partial responders andnon-responders is set equal to or up to 5% above the mRNA level of thehighest mRNA responder. The determination of whether partial respondersshould be administered a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof will depend upon the judgment ofthe treating physician and/or approval by a regulatory agency.

Methods that can be used to quantify specific RNA sequences in abiological sample are known in the art and include, but are not limitedto, fluorescent hybridization such as utilized in services and productsprovided by NanoString Technologies, array based technology(Affymetrix), reverse transcriptase qPCR as with SYBR® Green (LifeTechnologies) or TaqMan® technology (Life Technologies), RNA sequencing(e.g., RNA-seq), RNA hybridization and signal amplification as utilizedwith RNAscope® (Advanced Cell Diagnostics), or Northern blot. In somecases, mRNA expression values for various genes in various cell typesare publicly available (see, e.g., broadinstitute.org/ccle; andBarretina et al., Nature, 483:603-607, 2012).

In some embodiments, the state of a biomarker (as assessed, for example,by the level of RNA transcripts) in both the test biological sample(i.e., from the patient) and the reference standard or all members of apopulation is normalized before comparison. Normalization involvesadjusting the determined level of an RNA transcript by comparison toeither another RNA transcript that is native to and present atequivalent levels in both of the cells (e.g., GADPH mRNA, 18S RNA), orto a fixed level of exogenous RNA that is “spiked” into samples of eachof the cells prior to super-enhancer strength determination (Lovén etal., Cell, 151(3):476-82, 2012; Kanno et al., BMC Genomics 7:64, 2006;Van de Peppel et al., EMBO Rep., 4:387-93, 2003).

A patient (e.g., a human) suffering from a cancer described herein andidentified as described herein based on biomarker status may have beendetermined to be resistant (or to be acquiring resistance after someinitial efficacy) to a therapeutic agent that was administered prior tothe compound of Formula (I), (Ia), a species thereof, or a specifiedform thereof. For example, the cancer may be resistant or refractory toa chemotherapeutic agent, e.g., a Bcl-2 inhibitor such as venetoclax, aBET inhibitor, a CDK4/6 inhibitor such as palbociclib or ribociclib, aCDK9 inhibitor such as alvocidib, a FLT3 inhibitor, a MEK inhibitor sucha trametinib, a PARP inhibitor, such as olaparib or niraparib, a PI3Kinhibitor, such as alpelisib or capecitabine, a platinum-basedtherapeutic agent such as cisplatin, oxaliplatin, nedaplatin,carboplatin, phenanthriplatin, picoplatin, satraplatin (JM216), ortriplatin tetranitrate, a SERM, such as tamoxifen faloxifene, ortoremifene, or a steroid receptor degrading agent (e.g., a SERD, such asfulvestrant). Combination therapies including one or more of theseagents are also within the scope of the invention and are discussedfurther herein. For example, in one embodiment, the methods encompassthe use of or administration of a compound of Formula (I), (Ia), aspecies thereof or a specified form thereof, in combination with a SERD,such as fulvestrant, to treat a cancer (e.g., a breast cancer (e.g., anER+ breast cancer)) resistant to treatment with a CDK4/6 inhibitor suchas palbociclib or ribociclib.

In some embodiments, the prior therapeutic agent may be a platinum-basedanti-cancer agent administered as a monotherapy or in combination with aSOC agent. Most cancer patients eventually develop resistance toplatinum-based therapies by one or more of the following mechanisms: (i)molecular alterations in cell membrane transport proteins decreaseuptake of the platinum agent; (ii) molecular alterations in apoptoticsignaling pathways that prevent a cell from inducing cell death; (iii)molecular alterations of certain genes (e.g. BRCA1/2, CHEK1, CHEK2,RAD51) that restore the ability of the cell to repair platinumagent-induced DNA damage. K. N. Yamamoto et al., 2014, PloS ONE9(8):e105724. The term “molecular alterations” includes increased ordecreased mRNA expression from the genes involved in these functions;increased or decreased expression of protein from such genes; andmutations in the mRNA/proteins expressed from those genes.

Resistance is typically determined by disease progression (e.g., anincrease in tumor size and/or numbers) during treatment or a decrease inthe rate of shrinkage of a tumor. In some instances, a patient will beconsidered to have become resistant to a platinum-based agent when thepatient's cancer responds or stabilizes while on treatment, but whichprogresses within 1-6 months following treatment with the agent.Resistance can occur after any number of treatments with platinumagents. In some instances, disease progression occurs during, or within1 month of completing treatment. In this case, the patient is consideredto have never demonstrated a response to the agent. This is alsoreferred to a being “refractory” to the treatment. Resistance may alsobe determined by a treating physician when the platinum agent is nolonger considered to be an effective treatment for the cancer.

In some embodiments, the patient is or has been determined to beresistant to treatment with a CDK4/6 inhibitor administered as amonotherapy or in combination with a SOC agent.

CDK4/6 inhibitors in cancer (e.g., HR⁺ breast cancer) are known to blockentry into S phase of the cell cycle by inducing G1 arrest. Resistanceto CDK4/6 inhibitors in cancer (e.g., HR⁺ metastatic breast cancer) hasbeen shown to be mediated, in part, by molecular alterations that: 1)enhance CDK4/6 activity, such as amplifications of CDK6, CCND1, or FGFR1(Formisano et al., SABCS 2017, Publication Number GS6-05; Cruz et al.,SABCS 2017 Publication Number PD4-05), or 2) reactivate cell cycle entrydownstream of CDK4/6, such as RB1 loss and CCNE1 amplification(Condorelli, Ann. Oncol., 2017 PMID: 29236940; Herrera-Abreu, CancerResearch 2016 PMID: 27020857).

Unlike platinum-based agents which are typically administered for aperiod of time followed by a period without treatment, CDK4/6inhibitors, such as palbociclib, ribociclib or abemaciclib, areadministered until disease progression is observed. In some instances, apatient will be considered to have become resistant to a CDK4/6inhibitor when the patient's cancer initially responds or stabilizeswhile on treatment, but which ultimately begins to progress while stillon treatment. In some instances, a patient will be considered to beresistant (or refractory) to treatment with a CDK4/6 inhibitor if thecancer progresses during treatment without demonstrating any significantresponse or stabilization. Resistance may also be determined by atreating physician when the CDK4/6 inhibitor is no longer considered tobe an effective treatment for the cancer.

In case of any doubt, any of the specified forms of a compound ofFormula (I), (Ia), or a species thereof can be included in apharmaceutical composition used or administered (e.g., in an effectiveamount (e.g., a therapeutically effective amount) according to a methodof the invention. Pharmaceutical compositions useful in the methods ofthe invention can be prepared by relevant methods known in the art ofpharmacology. In general, such preparatory methods include the steps ofbringing a compound described herein, including compounds of Formula(I), (Ia), a species thereof, or a specified form thereof (e.g., apharmaceutically acceptable salt, solvate, stereoisomer, tautomer, orisotopic form thereof) into association with a carrier and/or one ormore other active ingredients (e.g., a second agent described herein)and/or accessory ingredients, and then, if necessary and/or desirable,shaping and/or packaging the product into a desired single-dose ormulti-dose unit (e.g., for oral dosing). The accessory ingredient mayimprove the bioavailability of a compound of Formula (I), (Ia), aspecies thereof, or a specified form thereof, may reduce and/or modifyits metabolism, may inhibit its excretion, and/or may modify itsdistribution within the body (e.g., by targeting a diseased tissue(e.g., a tumor). The pharmaceutical compositions can be packaged invarious ways, including in bulk containers and as single unit doses(containing, e.g., discrete, predetermined amounts of the active agent)or a plurality thereof, and any such packaged or divided dosage formsare within the scope of the invention. The amount of the activeingredient can be equal to the amount constituting a unit dosage or aconvenient fraction of a dosage such as, for example, one-half orone-third of a dose.

Relative amounts of the active agent/ingredient, the pharmaceuticallyacceptable carrier(s), and/or any additional ingredients in apharmaceutical composition of the invention can vary, depending upon theidentity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administeredand the disease to be treated. By way of example, the composition maycomprise between about 0.1% and 99.9% (w/w or w/v) of an activeagent/ingredient.

Pharmaceutically acceptable carriers useful in the manufacture of thepharmaceutical compositions described herein are well known in the artof pharmaceutical formulation and include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Pharmaceutically acceptable carriersuseful in the manufacture of the pharmaceutical compositions describedherein include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Pharmaceutical compositions used as described herein may be administeredorally. Such orally acceptable dosage forms may be solid (e.g., acapsule, tablet, sachet, powder, granule, and orally dispersible film)or liquid (e.g., an ampoule, semi-solid, syrup, suspension, or solution(e.g., aqueous suspensions or dispersions and solutions). In the case oftablets, carriers commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, can also be included. Inthe case of capsules, useful diluents include lactose and driedcornstarch. When aqueous suspensions are formulated, the activeagent/ingredient can be combined with emulsifying and suspending agents.In any oral formulation, sweetening, flavoring or coloring agents mayalso be added. In any of the various embodiments described herein, anoral formulation can be formulated for immediate release orsustained/delayed release and may be coated or uncoated. A providedcomposition can also be micro-encapsulated.

Compositions suitable for buccal or sublingual administration includetablets, lozenges and pastilles. Formulations can also be prepared forsubcutaneous, intravenous, intramuscular, intraocular, intravitreal,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intraperitoneal intralesional and by intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, subcutaneously, intraperitoneally or intravenously.Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by one ofordinary skill in the art that such compositions are generally suitablefor administration to animals of all sorts. Modification ofpharmaceutical compositions suitable for administration to humans inorder to render the compositions suitable for administration to variousanimals is well understood, and the ordinarily skilled veterinarypharmacologist can design and/or perform such modification.

Compounds described herein are typically formulated in dosage unit form,e.g., single unit dosage form, for ease of administration and uniformityof dosage. The specific therapeutically or prophylactically effectivedose level for any particular subject or organism will depend upon avariety of factors including the disease being treated and the severityof the disorder; the activity of the specific active ingredientemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific activeingredient employed; the duration of the treatment; drugs used incombination or coincidental with the specific active ingredientemployed; and like factors well known in the medical arts.

The amount of a compound required to achieve an optimum clinical outcomecan vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects,cancer to be treated, identity of the particular compound(s) to beadministered, and mode of administration. The desired dosage can bedelivered two or three times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a compound foradministration one or more times a day (e.g., once) to a 70 kg adulthuman may comprise about 1-100 mg, about 1-50 mg, about 1-35 mg (e.g.,about 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 mg), about 2-20 mg, about3-15 mg or about 10-30 mg (e.g., 10-20 or 10-25 mg). Here, and whereverranges are referenced, the end points are included. The dosages providedin this disclosure can be scaled for patients of differing weights orbody surface and may be expressed per m² of the patient's body surface.In certain embodiments, compositions of the invention may beadministered once per day. The dosage of a compound of Formula (I),(Ia), a species thereof or a specified form thereof (e.g., a saltthereof) can be about 1-100 mg, about 1-50 mg, about 1-25 mg, about 2-20mg, about 5-15 mg, about 10-15 mg, or about 13-14 mg. In certainembodiments, a composition of the invention may be administered twiceper day. In some embodiments, the dosage of a compound of Formula I or asubgenus or species thereof for each administration is about 0.5 mg toabout 50 mg, about 0.5 mg to about 25 mg, about 0.5 mg to about 1 mg,about 1 mg to about 10 mg, about 1 mg to about 5 mg, about 3 mg to about5 mg, or about 4 mg to about 5 mg.

As noted, the invention provides pharmaceutical kits configured fortreating cancer that include a compound of Formula (I), (Ia), a speciesthereof, or a specified form thereof and, optionally, anadditional/second therapeutic agent (e.g., second and third agents)selected from the second/additional agents described herein. Forexample, the second/additional agent can be: (a) a Bcl-2 inhibitor ordual Bcl-2/BCL-xL inhibitor, (b) a CDK inhibitor (e.g., a CDK4/6, CDK7,or CDK9 inhibitor), (c) a Flt3 inhibitor, (d) a PARP inhibitor, (e) aBET inhibitor, (f) an aromatase inhibitor, (g) a SERM, SERD, or estrogensuppressant, (h) a MEK inhibitor, or (i) a PI3 kinase inhibitor, which,as noted, may be selected from those disclosed herein. The kit caninclude optional instructions for: (a) reconstituting (if necessary) acompound of Formula (I), (Ia), a species thereof, or a specified formthereof and/or the second therapeutic agent; (b) administering each ofthe compound of Formula (I), (Ia), a species thereof, or a specifiedform thereof and/or the second therapeutic agent; and/or (c) a list ofspecific cancers for which the kit is useful or diagnostic methods bywhich they may be determined. The kits can also include any type ofparaphernalia useful in administering the active agent(s) containedtherein (e.g., tubing, syringes, needles, sterile dressings, tape, andthe like). Such kits, whether configured to deliver a monotherapyconsisting of a compound of Formula (I), (Ia), a species thereof, or aspecified variant thereof, or a combination therapy including anadditional/second agent selected from any one of those described herein,find utility in the diagnostic and treatment methods described herein.In some instances, the first and second agents will be in separatevessels (e.g., with the first agent confined to a first container andthe second agent confined to a second container) and/or formulated in apharmaceutically acceptable composition, optionally in unit dosage form,that includes the first agent, the second agent, and a pharmaceuticallyacceptable carrier. In some instances, the kits include a written insertor label with instructions to use the two (or more) therapeutic agentsin a patient suffering from a cancer (e.g., as described herein) andidentified as amenable to treatment by a method described herein. Theinstructions may be adhered or otherwise attached to a vessel or vesselscomprising the therapeutic agents. Alternatively, the instructions andthe vessel(s) can be separate from one another but present together in asingle kit, package, box, bag, or other type of container.Alternatively, or in addition, the written instructions can specify anddirect the user to a website or other media. The instructions in the kitwill typically be mandated or recommended by a governmental agencyapproving the therapeutic use of the combination (e.g., in a patientpopulation identified as described herein). The instructions mayoptionally comprise dosing information for each therapeutic agent, thetypes of cancer for which treatment of the combination was approved ormay be prescribed, physicochemical information about each of thetherapeutics, pharmacokinetic information about each of thetherapeutics, drug-drug interaction information, or diagnosticinformation (e.g., based on a biomarker or a method of identifying apatient for treatment as described herein). The kits of the inventioncan also include reagents useful in the diagnostic methods describedherein.

EXAMPLES

The compounds described herein can be prepared from readily availablestarting materials and according to the synthetic protocols describedbelow. Alternatively, one of ordinary skill in the art may readilymodify the disclosed protocols. For example, it will be appreciated thatwhere process conditions (e.g., reaction temperatures, reaction times,mole ratios of reactants, solvents, pressures, etc.) are given, otherprocess conditions can also be used. Additionally, and as will beapparent to one of ordinary skill in the art, protecting groups may beused to prevent certain functional groups from undergoing undesiredreactions. The choice of a suitable protecting group for a particularfunctional group as well as suitable conditions for protection anddeprotection are well known in the art. For example, numerous protectinggroups and guidance for their introduction and removal are disclosed byGreene et al. (Protecting Groups in Organic Synthesis, Second Edition,Wiley, New York, 1991, and references cited therein).

Also included within the Examples below are studies demonstrating thatdaily oral dosing of Compound 101 can induce dose-dependent TGI inovarian and breast tumor xenografts, with tumor regression observed atdoses as low as one-fifth of MTD. We also observed Compound 101 plasmaexposures that were dose proportional without accumulation upon repeateddialing dosing at therapeutic doses in mice (1-6 mg/kg). Compound 101induced rapid (4 hours) and sustained (24 hours) dose-dependentpharmacodynamic responses in xenograft tumor tissue that correlated withTGI, supporting but not mandating a QD dosing regimen. We also observedtumor regressions that were sustained after treatment with Compound 101was discontinued, at well-tolerated doses in multiple PDX models fromSCLC, TNBC, and HGSOC. Sustained regressions were associated with RBpathway alterations. In a study of combination therapy, Compound 101induced robust anti-tumor activity in combination with fulvestrant intreatment-resistant PDX models of ER+ breast cancer. Collectively, thesestudies highlight the broad potential for compounds of the invention ina variety of solid tumor types.

Example 1: Synthesis of Benzyl (2R,5R)-5-amino-2-methyl-piperidine-1-carboxylate and benzyl (2S,5S)-5-amino-2-methyl-piperidine-1-carboxylate Step 1: Benzyl5-(tert-butoxycarbonylamino)-2-methyl-piperidine-1-carboxylate

To a solution containing commercially available racemic trans tert-butylN-(6-methyl-3-piperidyl)carbamate (5 g, 23.33 mmol, 1 eq,) and NaHCO₃(13.72 g, 163.32 mmol, 7 eq) in tetrahydrofuran (THF; 50 mL) and H₂O (50mL), we added CbzCl (5.97 g, 35.00 mmol, 4.98 mL, 1.5 eq) dropwise at 0°C. The mixture was stirred at 15° C. for 2 hours then poured into water(50 mL) and extracted with ethyl acetate (EtOAc; 50 mL×3). The combinedorganic layer was washed with brine (50 mL×3), dried over Na₂SO₄, andfiltered. The filtrate was concentrated under reduced pressure, and theresidue was purified by medium pressure liquid chromatography (MPLC;SiO₂, PE:EtOAc=5:1 to 1:1) to give the title compound as a yellow solid(9.7 g, 18.04 mmol, 77.32% yield, 64.8% purity).

Step 2: Benzyl (2R, 5R)-5-amino-2-methyl-piperidine-1-carboxylate andbenzyl (2S, 5S)-5-amino-2-methyl-piperidine-1-carboxylate

To a mixture of racemic trans benzyl5-(tert-butoxycarbonylamino)-2-methyl-piperidine-1-carboxylate (9.7 g,27.84 mmol, 1 eq) in EtOAc (100 mL) we added HCl/EtOAc (15 mL, 4 M), andthe mixture was stirred at 15° C. for 1 hour. We then filtered themixture and collected the filter cake. The solid was dissolved inmethanol (MeOH; 15 mL) and the pH was adjusted to 9 using a stronglyacidic cation exchange resin (here, AMBERLYST® A21) before the mixturewas filtered and the filtrate was concentrated. The residue wasseparated by supercritical fluid chromatography (SFC; column: marketedby Daicel as CHIRALCEL® (chemicals for use in chromatography) ODH (250mm×30 mm, 5 μm); mobile phase: [0.1% NH₃.H₂O MeOH]; B %: 28%-28%, 16min) to afford title compound 1 (1.9 g, SFC: Rt=2.264 min, 93.2% ee,peak 1) and title compound 2 (1.9 g, SFC: Rt=2.593 min, 98.6% ee, peak2), both as light yellow solids. Peak 1 is structure 3. Peak 2 isstructure 4.

Example 2: Synthesis of 7-dimethylphosphoryl-3-[2-[[(3S,6S)-6-methyl-3-piper-idyl]amino]-5-(trifluoromethyl)pyrimidin-4-yl]-1H-indole-6-carbonitrile(Compound 100) Step 1: Benzyl (2S,5S)-5-[[4-(7-chloro-6-cyano-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl] amino]-2-methyl-piperidine-1-carboxylate

We stirred a mixture of7-chloro-3-[2-chloro-5-(trifluoromethyl)pyrimidin-4-yl]-1H-indole-6-carbonitrile(0.81 g, 2.27 mmol, 1 eq), benzyl(2S,5S)-5-amino-2-methyl-piperidine-1-carboxylate (732.20 mg, 2.95 mmol,1.3 eq) and N,N-diisopropylethylamine (DIEA or DIPEA; 879.41 mg, 6.80mmol, 1.19 mL, 3 eq) in N-methyl-2-pyrrolidone (NMP; 8 mL) at 140° C.for 1 hour. The reaction mixture was diluted with H₂O (100 mL) andextracted with EtOAc (50 mL×2). The combined organic layers were washedwith brine (100 mL×2), dried over Na₂SO₄, filtered, and concentratedunder reduced pressure to give a residue that was purified by columnchromatography (SiO₂, petroleum ether/ethyl acetate=10:1 to 4:1) toafford title compound as a yellow solid (1.1 g).

Step 2: Benzyl (2S,5S)-5-[[4-(6-cyano-7-dimethylphosphoryl-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-2-methyl-piperidine-1-carboxylate

We prepared a mixture of benzyl(2S,5S)-5-[[4-(7-chloro-6-cyano-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-2-methyl-piperidine-1-carboxylate (1.05 g, 1.85mmol, 1 eq), methylphosphonoylmethane (720.17 mg, 9.23 mmol, 5 eq),K₃PO₄ (783.45 mg, 3.69 mmol, 2 eq), Pd(OAc)₂ (41.43 mg, 184.54 μmol, 0.1eq), xantphos (C₃₉H₃₂OP₂; 106.78 mg, 184.54 μmol, 0.1 eq) anddimethylformamide (DMF; 10 mL) in a microwave sealed tube, degassed it,and purged it with N₂ (×3). The mixture was then stirred at 150° C. for1 hour in microwave. The reaction mixture was diluted with H₂O (100 mL)and extracted with ethyl acetate (EtOAc; 50 mL×3). The combined organiclayers were washed with brine (150 mL×2), dried over Na₂SO₄, filtered,and concentrated under reduced pressure to give a residue that wepurified by column chromatography (SiO₂, petroleum ether/ethylacetate=10:1 to 1:1) to afford the title compound as a yellow oil (490mg).

Step 3: 7-dimethylphosphoryl-3-[2-[[(3S,6S)-6-methyl-3-piperidyl]amino]-5-(trifluoromethyl)pyrimidin-4-yl]-1H-indole-6-carbonitrile

To a solution ofbenzyl(2S,5S)-5-[[4-(6-cyano-7-dimethylphosphoryl-1H-indol-3-yl)-5-(trifluoromethyl)pyrimidin-2-yl]amino]-2-methyl-piperidine-1-carboxylate(440 mg, 720.64 μmol, 1 eq) in EtOAc (5 mL), we added Pd/C (200 mg, 10%purity) under N₂. We degassed the suspension under vacuum, purged itwith H₂ several times, then stirred the mixture under H₂ (15 psi) at 20°C. for 3 hours before filtering it. The filtrate was concentrated togive a residue we purified by prep-HPLC (high performance liquidchromatography; neutral condition) to yield the title compound as awhite solid (142.2 mg).

The reaction was combined with another reaction in 50 mg scale forpurification by liquid chromatography mass spectrometry (LCMS). LCMS:ET6034-1492-P1C: (M+H⁺): 477.1 @2.572 (10-80% ACN (acetonitrile) in H₂O4.5 minutes). ¹H NMR (400 MHz, DMSO (dimethylsulfoxide)-d6) δ 8.74 (brd, J=7.89 Hz, 1H), 8.65-8.44 (m, 2H), 8.17 (br d, J=15.35 Hz, 1H), 7.84(brt, J=8.11 Hz, 1H), 7.67 (brt, J=7.02 Hz, 1H), 3.81 (br s, 1H), 3.10(br d, J=11.40 Hz, 1H), 2.45-2.38 (m, 1H), 2.02 (d, J=13.59 Hz, 8H),1.64 (br d, J=11.40 Hz, 1H), 1.49-1.34 (m, 1H), 1.11 (br d, J=10.96 Hz,1H), 0.97 (br d, J=5.70 Hz, 3H).

Example 3: Synthesis of (S)-6,6-dimethylpiperidin-3-amine

We dissolved (S)-tert-butyl (6-oxopiperidin-3-yl)carbamate (1.00 g, 4.67mmol) (Tetrahedron Letters, 36:8205, 1995) in THF (47 mL) and cooled thesolution to −10° C. Zirconium (IV) chloride (2.61 g, 11.22 mmol) wasadded, and the mixture was stirred for 30 minutes at this temperature. Amethylmagnesium bromide solution (3M in ether, 20.25 mL, 60.75 mmol) wasadded, and the reaction mixture was allowed to slowly warm up to roomtemperature, at which it was stirred overnight. The solution wasquenched with 30% aqueous NaOH, diluted with EtOAc, filtered, and thenextracted 3 times with EtOAc. The organics were combined, dried oversodium sulfate, filtered, and concentrated in vacuo to provide the crudeproduct as a yellow oil that was used without purification. The oil wasdissolved in dichloromethane (DCM; 47 mL) and trifluoroacetic acid (TFA;3.58 mL, 46.73 mmol) was added. We stirred the reaction mixture at roomtemperature for 16 hours, concentrated it in vacuo and co-evaporated ita few times with DCM to provide the crude title compound as a brown oil,which we used in the next step without further purification.

Example 4: Synthesis of(S)-7-(dimethylphosphoryl)-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile(Compound 101) Step 1: 7-Bromo-1H-indole-6-carboxylic Acid

We stirred a solution of vinylmagnesium bromide (1.0 M in THF (159 mL,159 mmol) at −78° C. and added to it, dropwise, over a period of 1 hour,a solution of 2-bromo-3-nitrobenzoic acid (10.0 g, 39.8 mmol) in THF(159 mL). The reaction mixture was allowed to reach room temperature andwas stirred at that temperature overnight. The reaction mixture was thenpoured over saturated aqueous ammonium chloride (150 mL) and acidifiedto a pH 2, using aqeous 1M HCl. We extracted the crude product withEtOAc (3×200 mL), dried the extract over sodium sulfate, filtered it,and concentrated it in vacuo. The residue was then triturated in DCM(100 mL) and dried overnight with a flow of air to provide the titlecompound as a light brown solid (7.58 g, 31.58 mmol, 79% yield).

Step 2: 7-Bromo-1H-indole-6-carboxamide

We stirred a solution of 7-bromo-1H-indole-6-carboxylic acid (6.58 g,27.4 mmol) in DMF (54.8 mL) at 0° C. and added 1,1′-carbonyldiimidazole(CDI; 8.89 g. 54.8 mmol) to it portion wise. The mixture was stirred for5 minutes, and the intermediate was observed by LCMS. We then addedNH₄OH (39.5 mL, 274 mmol) at 0° C., and the solution was stirred for 5minutes. The reaction was quenched with saturated aqueous ammoniumchloride (25 mL) and saturated aqueous sodium chloride (25 mL) thendiluted with 2-methyltetrahydroftiran (MeTHF; 50 mL). We separated thephases and washed the organic layer again with saturated aqueousammonium chloride (25 mL) and saturated aqueous sodium chloride (25 mL).The organic layer was then dried over sodium sulfate, filtered, andconcentrated in vacuo to provide the title compound, which was carriedover to the next step assuming the quantitative yield.

Step 3: 7-Bromo-1H-indole-6-carbonitrile

We added Et₃N (triethylamine; 44.1 mL, 315 mmol) to a suspension of7-bromo-1H-indole-6-carboxamide (7.53 g, 31.5 mmol) in DCM (315 mL) at0° C. and stirred the resulting orange solution at that temperatureuntil we obtained a homogeneous solution. MsCl (12.2 mL, 157 mmol) wasthen added dropwise, and the solution was stirred at 0° C. for 5minutes. We diluted the mixture with ethyl acetate and washed it withsaturated aqueous sodium bicarbonate before extracting the aqueous layertwice more with ethyl acetate. The organic layers were combined, washedwith brine, dried over sodium sulfate, filtered, and concentrated invacuo. The residue was purified by filtering it through a pad of silica(eluting with ethyl acetate) to provide the title compound as a brownsolid (5.80 g, 26.24 mmol, 83% yield).

Step 4:7-Bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile

We added AlCl₃ (1.83 g, 13.6 mmol) to a solution of2,4-dichloro-5-trifluoromethylpyrim-idine (3.66 mL, 27.2 mmol) in1,2-dichloroethane (DCE; 36.2 mL) and stirred the resulting suspensionat 80° C. for 30 minutes. We added 7-bromo-1H-indole-6-carbonitrile(2.00 g, 9.05 mmol) to the mixture and stirred the resulting redsolution at 80° C. until full conversion (4 hours). The reaction mixturewas then diluted with MeTHF (100 mL) and washed with water (100 mL). Theaqueous layer was extracted with 2-MeTHF (100 mL), and the organicextracts were combined, dried over sodium sulfate, filtered, andconcentrated in vacuo. Formation of two possible regioisomers wasobserved in a ratio of 3:1 (desired/undesired). We purified the residueby reverse phase chromatography on C18 (MeCN (acetonitrile) in water, 15to 80% gradient) to provide the title compound as a beige solid (1.51 g,3.76 mmol, 42% yield). ¹H NMR (500 MHz, DMSO) δ 13.00 (brs, 1H), 9.17(s, 1H), 8.35 (d, J=8.4 Hz, 1H), 8.16 (d, J=2.6 Hz, 1H), 7.71 (d, J=8.4Hz, 1H).

Step 5:(S)-7-Bromo-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile

We dissolved7-bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile(200 mg, 0.498 mmol), (S)-6,6-dimethylpiperidin-3-amine (95.8 mg, 0.747mmol), and DIPEA (174 μL, 0.996 mmol) in NMP (4 mL) then stirred thereaction mixture at 130° C. in an oil bath until full conversion (3hours). The mixture Was cooled to room temperature, loaded directly ontoa C18 column and purified by reverse phase chromatography (MeCN with0.1% FA (formic acid) in water also containing 0.1% FA, 0 to 100%gradient). The title compound was obtained as a beige solid (245 mg.0497 mmol, quantitative yield).

Step 6:(S)-7-(dimethylphosphoryl)-3-(2-((6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)-pyrimidin-4-yl)-1H-indole-6-carbonitrile

We combined(S)-7-bromo-3-(2-((6,6-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)-pyrimidin-4-yl)-1H-indole-6-carbonitrile(180.0 mg, 0.365 mmol), Xantphos (21.5 trig, 36.5 μmol), palladium (II)acetate (4.14 mg 18.2 μmol), and K₃PO₄ (85.2 mg. 0.401 mmol) in a 2.5 mLmicrowave vial under nitrogen. Dimethylphosphine oxide (73 mg, 0.912mmol) was dissolved in anhydrous DMF (1 mL), and the solution wasdegassed before combining with the other reactants in a microwave vial.The sealed vial with the reaction mixture was then submitted to heat ina microwave reactor at 150° C. for 45 minutes. The reaction mixture wascooled to room temperature, loaded directly onto a C18 column, andpurified by reverse phase chromatography (MeCN in aqueous 10 mM ammoniumformate pH 3.8, 15 to 35% gradient). The title compound was obtained asan off-white solid (76 mg, 0.155 mmol, 42% yield).

Example 5: Synthesis of (3S)-1-benzyl-5, 5-dimethyl-piperidin-3-amineStep 1: Methyl (2S)-5-oxopyrrolidine-2-carboxylate

We added SOCl₂ (215.62 g, 1.81 mol, 131.47 mL, 2 eq) to a solution of(2S)-5-oxopyrrolidine-2-carboxylic acid (117 g, 906.18 mmol, 1 eq) inMeOH (500 mL) at 0° C. The mixture was stirred at 18° C. for 1 hourbefore the reaction mixture was concentrated. We diluted the residuewith EtOAc (1000 mL) and TEA (triethylamine; 150 mL) and filtered thesolid that was formed. The filtrate was evaporated to afford the titlecompound as a light yellow oil (147 g, crude) to be used directly in thenext step without any further purification.

Step 2: (S)-1-tert-butyl 2-methyl 5-oxopyrrolidine-1,2-dicarboxylate

To a solution of methyl (2S)-5-oxopyrrolidine-2-carboxylate (147 g, 1.03mol, 1 eq), DMAP (4-dimethylaminopyridine; 15.06 g, 123.24 mmol, 0.12eq) and TEA (259.80 g, 2.57 mol, 357.35 mL, 2.5 eq) in EtOAc (500 mL) weadded tert-butoxycarbonyl tert-butyl carbonate (291.37 g, 1.34 mol,306.71 mL, 1.3 eq), dropwise, at 0° C. The mixture was then stirred at20° C. for 16 hours. We then washed the reaction mixture with HCl (0.5M, 1000 mL), saturated NaHCO₃ (1000 mL), brine (1500 mL), dried it overNa₂SO₄, and filtered and concentrated it under reduced pressure to givea residue that was then purified by re-crystallization from methyltert-butyl ether (MTBE; 250 mL). The reaction mixture was filtered andevaporated to afford the title compound as a white solid (2 batchesobtained; Batch 1: 108 g, 100% HPLC purity; Batch 2: 53 g, 90% ¹H NMRpurity).

Step 3: (S)-1-tert-butyl 2-methyl4,4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate

We added LiHMDS (lithium hexamethyldisilazide; 1 M, 172.66 mL, 2.1 eq),dropwise, to a solution of (S)-1-tert-butyl 2-methyl 5-oxopyrrolidine-1,2-dicarboxylate (20 g, 82.22 mmol, 1 eq) in THF (500 mL) at −78° C.under N₂ atmosphere. After addition, the mixture was stirred at thattemperature for 30 minutes before we added CH₃I (35.01 g, 246.65 mmol,15.36 mL, 3 eq), dropwise, at −78° C. under N₂ atmosphere. The resultingmixture was stirred at 20° C. for 2.5 hours. The reaction mixture wasdiluted with saturated aqueous NH₄Cl (1000 mL) and extracted with EtOAc(300 mL×3). The combined organic layers were washed with brine (500 mL),dried over Na₂SO₄, filtered, and concentrated under reduced pressure togive a residue that was purified by MPLC (SiO₂, PE:EtOAc=4:1-3:1) toafford the title compound as a light yellow solid (8 g, 25.95 mmol,31.56% yield, 88% purity).

Step 4: tert-butylN-[(1S)-4-hydroxy-1-(hydroxymethyl)-3,3-dimethyl-butyl]carbamate

To a solution of (S)-1-tert-butyl 2-methyl 4,4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate (4.3 g, 15.85 mmol, 1 eq)in THF (35 mL) we added NaBH₄ (1.80 g, 47.55 mmol, 3 eq), by portions,at 0° C. under N₂. After addition, EtOH (ethanol; 8.25 g, 179.09 mmol,10.47 mL, 11.3 eq) was added dropwise at 0° C. The resulting mixture wasstirred at 20° C. for 16 hours then poured into saturated aqueous NH₄Cl(250 mL) and extracted with EtOAc (100 mL×3). The combined organiclayers were washed with brine (250 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to afford the title compound as acolorless oil (3.67 g, crude), which was used directly in the next stepwithout any further purification

Step 5:[(2S)-2-(tert-butoxycarbonylamino)-4,4-dimethyl-5-methylsulfonyloxy-pentyl]methanesulfonate

To a solution of tert-butylN-[(1S)-4-hydroxy-1-(hydroxymethyl)-3,3-dimethyl-butyl]carbamate (3.67g, 14.84 mmol, 1 eq) and TEA (6.01 g, 59.35 mmol, 8.26 mL, 4 eq) inEtOAc (25 mL) we added methanesulfonyl chloride (5.10 g, 44.52 mmol,3.45 mL, 3 eq), dropwise, at 0° C. The resulting mixture was stirred at20° C. for 12 hours then poured into H₂O (200 mL). EtOAc (50 mL×3) wasused to extract the product. The organic layer was washed with brine (30mL), dried over Na₂SO₄, filtered and evaporated to afford the titlecompound as a colorless oil (6.06 g crude) that was used directly in thenext step without any further purification.

Step 6: Tert-butyl N-[(3S)-1-benzyl-5,5-dimethyl-3-piperidyl] carbamate

A flask was fitted with [(2S)-2-(tert-butoxycarbonylamino)-4,4-dimethyl-5-methyl-sulfonyloxypentyl] methanesulfonate (6.06 g, 15.02mmol, 1 eq), phenylmethanamine (5.15 g, 48.06 mmol, 5.24 mL, 3.2 eq) anddimethoxyethane (DME; 50 mL). We heated the reaction mixture to 70° C.for 16 hours then poured it into H₂O (40 mL). DCM (40 mL×3) was used toextract the product. The organic layer was washed with brine (30 mL),dried over Na₂SO₄, filtered and evaporated to afford the crude product,which was purified twice by MPLC (SiO₂, PE:EtOAc=20:1-10:1) to affordthe title compound as a colorless oil (580 mg, 1.49 mmol, 9.91% yield,81.7% purity).

Step 7: (3S)-1-benzyl-5, 5-dimethyl-piperidin-3-amine

A flask was fitted with tert-butyl N-[(3S)-1-benzyl-5,5-dimethyl-3-piperidyl]carbamate (300 mg, 942.05 μmol, 1 eq) inHCl/EtOAc (15 mL). The mixture was stirred at 25° C. for 1 hour, afterwhich some white precipitate formed. We filtered the mixture, and thecake was washed by EtOAc (5 mL), collected and dried over vacuum toafford the title compound as a white solid (220 mg, 738.23 μmol, 78.36%yield, 85.5% purity, HCl) as a white solid to be used directly in thenext step.

Example 6: Synthesis of(S)-7-(dimethylphosphoryl)-3-(2-((5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile(Compound 102) Step 1:(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-7-bromo-1H-indole-6-carbonitrile

We dissolved7-bromo-3-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-H-indole-6-carbonitrile(168 mg, 0.418 mmol), (S)-1-benzyl-5,5-dimethylpiperidin-3-amine (128mg, 0.585 mmol), and DIPEA (221 μL, 1.26 mmol) in NMP (2 mL). We stirredthe reaction mixture at 130° C. in an oil bath until full conversion (4hours). The mixture was cooled to room temperature, diluted with EtOAcand washed with saturated aqueous LiCl. The organic layer was separated,dried over sodium sulfate, filtered, and concentrated in vacuo toprovide the crude title compound (240 mg, 0.411 mmol, quant. yield),which was used in the next step without further purification.

Step 2:(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-7-(dimethylphosphoryl)-1H-indole-6-carbonitrile

We combined(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoro-methyl)pyrimidin-4-yl)-7-bromo-1H-indole-6-carbonitrile(240 mg, 0.411 mmol), Xantphos (24.3 mg, 41.1 μmol), palladium (II)acetate (4.66 mg, 20.6 μmol), and K₃P0₄ (96.0 mg, 0.452 mmol) in a 2.5mL microwave vial under nitrogen. Dimethylphosphine oxide (39.2 mg,0.494 mmol) was dissolved in anhydrous DMF (1 mL), and the solution wasdegassed before combining with the other reactants in a microwave vial.The sealed vial with the reaction mixture was then submitted to heat ina microwave reactor at 145° C. for 45 minutes. The reaction mixture wasthen cooled to room temperature, diluted with 2-MeTHF and washed withsaturated aqueous NaHCO₃ and brine. The organic layer was separated,dried over sodium sulfate, filtered, and concentrated in vacuo beforethe residue was purified by reverse phase chromatography on C18 (MeCN inaqueous 10 mM ammonium formate pH 3.8, 0 to 100% (gradient). The titlecompound was obtained as a pale brown oil (58.0 mg, 0.10 mmol, 24%yield).

Step 3:(S)-7-(dimethylphosphoryl)-3-(2-((5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-1H-indole-6-carbonitrile

Under a nitrogen atmosphere, to a stirring solution of(S)-3-(2-((1-benzyl-5,5-dimethylpiperidin-3-yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)-7-(dimethylphosphoryl)-1H-indole-6-carbonitrile(58.0 mg, 0.10 mmol) in EtOH (12.5 mL), we added Pd/C 10% w/w (1.1 mg,0.01 mmol) and Boc₂O (di-t-butyl decarbonate; 65.5 mg, 0.30 mmol). Thereaction mixture was evacuated and back-filled with nitrogen (×3) beforebeing filled with hydrogen. The reaction mixture was then stirred atroom temperature overnight under hydrogen atmosphere. After 16 hours, weobserved an incomplete conversion and therefore filtered the reactionmixture through a pad of CELITE® and concentrated it under reducedpressure. The reaction was then repeated with the residue as describedabove. After almost complete consumption of starting material (48hours), the reaction mixture was filtered through a pad of CELITE® andconcentrated in vacuo to provide the crude product, which was engaged inthe next step. Thus, the obtained oil was re-dissolved in DCM (5 mL),and TFA (0.23 mL, 3.0 mmol) was added. The reaction mixture was stirredat room temperature overnight. The mixture was then concentrated invacuo, and the residue was purified by reverse phase chromatography onC18 (MeCN in aqueous 10 mM ammonium formate pH 3.8, 0 to 100% gradient)to provide the title compound as a white solid (11.11 mg, 0.023 mmol,23% yield over two steps).

Example 7: Inhibition of CDK Kinase Activity

We assayed some compounds for inhibition of CDK7, CDK9, CDK12, and CDK2activity at Biortus Biosciences (Jiangyin, Jiangsu Province, P.R. ofChina) using kinase assays for each CDK developed with a Caliper/LabChipEZ Reader (Perkin Elmer, Waltham, Mass.). These assays measure theamount of phosphorylated peptide substrate produced as a fraction of thetotal peptide following an incubation period at 27° C. with thefollowing components: test compounds (variable concentrations from 10 μMdown to 0.508 nM in a series of 3-fold serial dilutions), active CDKprotein (with the indicated cyclin, listed below for each CDK), ATP (ateither the K_(m) concentrations listed below for each CDK/cyclin or 2 mMATP), and substrate peptide (listed below) in the following buffer:2-(N-morpholino)ethanesulfonate (MES buffer, 20 mM), pH 6.75, 0.01%(v/v) Tween 20 detergent, 0.05 mg/mL bovine serum albumin (BSA), and 2%DMSO.

Specifically, the CDK7 inhibition assay used CDK7/Cyclin H/MAT1 complex(6 nM) and “5-FAM-CDK7tide” peptide substrate (2 μM, synthesizedfluorophore-labeled peptide with the sequence5-FAM-YSPTSPSYSPTSPSYSPTSPSKKKK (SEQ ID NO:1), where “5-FAM” is5-carboxyfluorescein) with 6 mM MgCl2 in the buffer composition listedabove where the apparent ATP K_(m) for CDK7/Cyclin H/MAT1 under theseconditions is 50 μM. The CDK9 inhibition assay used CDK9/Cyclin T1complex (8 nM) and “5-FAM-CDK9tide” peptide substrate (2 μM, synthesizedfluorophore-labeled peptide with the sequence: 5-FAM-GSRTPMY-NH₂ (SEQ IDNO:2), where 5-FAM is defined above and NH₂ signifies a C-terminal amidewith 10 mM MgCl₂ in the buffer composition listed above. The CDK12inhibition assay used CDK12 (aa686-1082)/Cyclin K complex (50 nM) and“5-FAM-CDK9tide” (2 μM) as defined above, with 2 mM MgCl₂ in the buffercomposition above. The CDK2 inhibition assay used CDK2/Cyclin E1 complex(0.5 nM) and “5-FAM-CDK7tide” (2 μM) as defined above, with 2 mM MgCl₂in the buffer composition listed above.

The incubation period at 27° C. for each CDK inhibition assay was chosensuch that the fraction of phosphorylated peptide product produced ineach assay, relative to the total peptide concentration, wasapproximately 20% (±5%) for the uninhibited kinase (35 minutes for CDK7,35 minutes for CDK2, 3 hours for CDK12, and 15 minutes for CDK9). Incases where the compound titrations were tested and resulted ininhibition of peptide product formation, these data were fit to producebest-fit IC₅₀ values. The best-fit IC₅₀ values at K_(m) ATP for eachCDK/Cyclin, except for CDK7/Cyclin H/MAT1, were used to calculate K_(i)values, or the apparent affinity of each inhibitor for each CDK/Cyclinfrom the kinase activity inhibition assay, according to theCheng-Prusoff relationship for ATP substrate-competitive inhibition(Cheng and Prusoff, Biochem. Pharmacol., 22(23):3099-3108, 1973), with acorrection term for inhibitor depletion due to the enzyme concentration(Copeland, “Evaluation of Enzyme Inhibitors in Drug Discover: A Guidefor Medicinal Chemists and Pharmacologists,” Second Edition, March,2013; ISBN: 978-1-118-48813-3):

${IC_{50}} = {{K_{i}( {1 + \frac{\lbrack{Substrate}\rbrack}{K_{m}}} )} + \frac{\lbrack{Enzyme}\rbrack}{2}}$

Due to tight-binding inhibition and the limits of the CDK7/Cyclin H/MAT1assay, instead of calculating the apparent K_(i) values for eachinhibitor, the K_(d), or direct compound binding affinity, was measuredusing surface plasmon resonance (SPR) as described below.

Example 8: CDK7/Cyclin H Surface Plasmon Resonance (SPR) Assay Method

We measured binding kinetics and affinities of selected compounds to theCDK7/Cyclin H dimer using a Biacore T200 surface plasmon resonance (SPR)instrument (GE Healthcare). The dimer was amine-coupled to a CM5 sensorchip at pH 6.5 in 10 mM MES buffer at a concentration of 12.5 μg/mL witha flow rate of 10 μL/min. Target protein was immobilized on two flowcells for 12-16 seconds to achieve immobilized protein levels of 200-400Response Units.

Compounds were titrated from 0.08-20 nM in a 9-step, 2-fold serialdilution in 10 mM HEPES buffer at pH 7.5 with 150 mM NaCl, 0.05%Surfactant P20, and 0.0002% DMSO. Each compound concentration cycle wasrun at 100 μL/min with 70 second contact time, 300 second dissociationtime, 60 second regeneration time with 10 mM glycine pH 9.5, and 400second stabilization time. For each compound, 0 nM compound controls andreference flow-cell binding were subtracted to remove background andnormalize data. Compound titrations were globally fit by Biacore T200Evaluation Software (GE Healthcare) using kinetics mode. Best-fit valuesfor compound binding on-rate (k_(on)) and dissociation off-rate(k_(off)) for CDK7/Cyclin H were determined and these values were usedto calculate the compound affinity (K_(d)) for CDK7/Cyclin H using thefollowing equation:

${{K_{d}(M)} = \frac{k_{off}( s^{- 1} )}{k_{on}( {M^{- 1}s^{- 1}} )}}.$

Compound selectivity for CDK7 over CDK2, CDK9, or CDK12 were determinedbased on the ratios of K_(i) values for the off-target CDKs relative tothe direct compound binding K_(d) for CDK7 measured by SPR according to:

${Selectivity} = {\frac{K_{i,{{off}\;{target}}}}{K_{d,{{CDK}\; 7}}}.}$

The inhibitory and dissociation constants and selectivity of theindicated compounds (three compounds of the invention and fourcomparators) against CDK2, CDK7, CDK9, and CDK12 are shown in the tableof FIG. 1. As can be seen, each of the compounds of the invention is atleast 1300-fold and up to 40,000-fold more specific for CDK7 than forthe other CDKs tested.

Example 9: Inhibition of Cell Proliferation (Compounds 100-102)

The HCC70 cell line was derived from human TNBC, and we testedrepresentative compounds of the invention, at different concentrations(from 4 μM to 126.4 μM; 0.5 log serial dilutions), for their ability toinhibit the proliferation of those cells. More specifically, we testedthe same compounds tested above for CDK7 selectivity (the structures ofwhich are shown in FIG. 1), and we used the known CDK inhibitorsdinaciclib (or N-((1S,3R)-3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)cyclohexyl)-5-((E)-4-(dimethylamino)but-2-enamido)picolinamide)and triptolide as positive controls. The cells were grown inATCC-formulated RPMI-1640 medium (ATCC 30-2001) supplemented with 10%fetal bovine serum (FBS), at 37° C. in a humidified chamber in thepresence of 5% CO₂. We conducted proliferation assays over a 72-hourtime period using a CyQUANT® Direct Cell Proliferation Assay (LifeTechnologies, Chicago, Ill. USA) according to the manufacturer'sdirections and utilizing the reagents supplied with the kit. The resultsof the assay are shown in the Table below.

Compound HCC70 EC₅₀ (nM) Compound 100 0.98 Compound 101 5.6 Compound 1022.1 Comparator 1 0.53 Comparator 2 260 Comparator 3 24 Comparator 4 110

Example 10: TGI in Patient-Derived Xenograft (PDX) Models

Tumor growth inhibition was evaluated in estrogen receptor-positivebreast cancer (ER+BC) PDX models selected in vivo for resistance to theCDK4/6 inhibitor palbociclib (ST1799, n=1) or resistance to bothpalbociclib and fulvestrant (ST941, n=1). Dosing was initiated whentumors were 100-200 mm³. Mice were treated with either Compound 101, QD(6 mg/kg, once daily, by mouth); fulvestrant, SC (2.5 mg/kg, once weeklydosing, by subcutaneous injection); palbociclib, QD (50 mpk, once daily,by mouth) or in combination of Compound 101 (6 mg/kg, once daily, bymouth) and fulvestrant (2.5 mg/kg, once weekly, by subcutaneousinjection) over the course of 28 days, followed by 21 days ofobservation. Tumor growth inhibition (TGI) was calculated on the lastday of dosing using the formula: TGI=(V_(c1)−V_(t1))/(V_(c0)−V_(t0)),where V_(c1) and V_(t1) are the mean volumes of control and treatedgroups at the time of tumor extraction, while V_(c0) and V_(t0) are thesame groups at the start of dosing.

In the palbociclib-resistant ER+BC PDX (ST1799) model, the combinationof Compound 101 and fulvestrant induced significant TGI (89%), with noevident tumor regrowth up to 21 days after dosing cessation,distinguishing the observed effects from Compound 101 (83%), fulvestrant(60%) or palbociclib (21%) when administered as single agents.Additionally, the combination of Compound 101 and fulvestrant wassuperior to the SOC combination of palbociblib and fulvestrant (75%). Ina palbociclib and fulvestrant double-resistant ER+BC PDX model (ST941),Compound 101 administered as a single agent resulted in 33% TGI andfulvestrant and palbociclib as single agents or fulvestrant andpalbociclib in combination had no activity. In contrast, the combinationof Compound 101 and fulvestrant demonstrated significant TGI (68%;p<0.0001 vs fulvestrant as a single agent), suggesting re-sensitizationto fulvestrant.

FIG. 2 illustrates the TGI results from the palbociclib resistant HR+BCPDX model ST1799, and FIG. 3 illustrates the TGI results from thepalbociclib and fulvestrant resistant HR+BC PDX model ST941. We alsoobserved TGI in four additional PDX models; BR5010 (modeling TNBC),LU5178 (modeling small cell lung cancer (SCLC)), OV15398 (modeling highgrade serous ovarian cancer (HGSOC)), and ST390 (modeling pancreaticductal adenocarcinoma (PDAC)). In the TNBC model, Compound 101 wasorally administered to tumor-bearing NOD/SCID mice at 10 mg/kg QD or 5mg/kg BID for 21 days. In the SCLC and HGSOC models, Compound 101 wasorally administered to tumor-bearing NOD/SCID mice at 3 mg/kg BID for 21days. In the PDAC model, Compound 101 was orally administered totumor-bearing NOD/SCID mice at 6 mg/kg QD. In the TNBC, SCLC, and HGSOCmodels, tumor volume was measured during the treatment period and for anadditional 21 days after treatment ceased. The % TGI observed at the endof treatment (day 21) was calculated as: 1−[(Mean TV Compound 101 @EOT−Mean TV Compound 101 @ Day 0)/(Mean TV Veh @ EOT−Mean TV Veh @ Day0)]×100. The % regression was calculated as: (Mean TV Compound 101 @EOT)/(Mean TV Compound 101 @ Day 0)×100. The same calculations were usedfor end of study (day 42). The results are shown in FIG. 4. Theseresults demonstrate deep and sustained TGI, including regressions, atwell tolerated doses, in a variety of tumor types. Dose-dependenttranscriptional responses in xenograft tissue were observed within 4hours of dosing and were sustained for 24 hours. Similar TGI was seenwhen the same total dose was administered either QD or BID in the TNBCPDX model, suggesting that the effect was AUC or C_(min) driven.Moreover, the TGI observed in SCLC (in the LU5178 PDX model) had notbeen observed in previous studies with a covalent CDK7 inhibitor (datanot shown). Regarding the model of PDAC, we found Compound 101 induced100% TGI over the time examined (˜28 days) at a dose well below the MTD:at day 21, tumor volume was ˜1,250 mm³ in vehicle-treated mice but onlyabout 250 mm³ in Compound 1-treated mice (6 mg/kg QD, PO). WhileCompound 101 could achieve 100% TGI at sub-MTD doses in the tested PDACPDX tumors, a covalent CDK7 inhibitor achieved only modest TGI at itsMTD (40 mg/kg BIW, by IV administration, with evident body weight loss(8.4%) and necrosis at the injection site; data not shown).

Example 11. In Vitro Studies of Compound 101 in Combination with VariousSecond Agents

In the studies described here, cancer cell lines from HR+ breast cancers(lines T47D; PIK3CA p.H1047R, MCF7; PIK3CA p.E545K), SCLC, (NCI-H1048)and CRCs (lines RKO; BRAF p.V600E, SW480; KRAS p.G12V) were grown to 70%confluency in their media of preferences based on the manufacturerrecommendations. In the SCLC cell line (NCI-H1048), Compound 101 wastested in combination with SOC chemotherapy agents gemcitabine (a DNAsynthesis inhibitor) and carboplatin (a DNA damage agent). In a CRC cellline (RKO; BRAF p.V600E), Compound 101 was tested in combination withSOC chemotherapy agent oxaliplatin (a DNA damage agent). Additionally,in CRC, Compound 101 was tested in combination with the selective MAPKpathway inhibitor trametinib in two CRC cell lines harboring MAPKpathway alterations; RKO (BRAF p.V600E mutant) and SW480 (KRAS p.G12Vmutant). Compound 101 was tested in combination with the SOC agentcapecitabine (an antimetabolite) in HR+MCF-7 cells. In the HR+ celllines MCF7 and T47D, which have activating mutations in the PIK3CAkinases, Compound 101 was tested in combination with the PIK3CAselective inhibitor alpelisib.

On the day of assay, cells were lifted and counted using the Countess IIFL (Life Technologies). Using an automated dispenser (here, Multidrop™Combi Reagent Dispenser), 50 μL of preferred cell media containing20,000-50,000 cells/ml was distributed into black 384-well Nunc plates(Thermo) and allowed to adhere overnight prior to compound addition.Compound arrays were distributed to 384 well assay plates using SynergyPlate Format with an HP D300e Digital Dispenser (HP). Compound 101 andother TEST agents were dissolved in DMSO to make a stock solution thatallowed for more accurate dispensing. However, due to solubility andreactivity, platinum agents were dissolved in water with an addition of0.03% Tween-20 to allow for dispensing with digital printer. Compoundswere plated in each quadrant of a 384-well plate in quadruplicate. Eachquadrant contained test wells with combination of SY-1365 andcarboplatin or oxaliplatin (TEST/test agent) as well as single agentcolumns, and vehicle wells.

Compound 101 was plated in across from left to right in a high to lowconcentration (8 columns), and the varying concentrations of carboplatinor oxaliplatin (TEST) plated in synergy wells from top to bottom (7rows). Concentrations were selected to cover the full isobologram ofactivity based on activity of single agents. Single agents were platedin dose in two columns, with a third separate column of justDMSO/vehicle treated wells. A separate plate for each cell line wasseeded to allow for determination of a “Time Zero”/“Day Zero” number ofcells to parse the differential cytostatic vs cytotoxic effects. On theday compounds were added, viability of the time zero plate wasdetermined to identify growth inhibition from cell killing effects.

After addition of compound, cell plates were incubated for 5 days in a37° C. incubator. Cell viability was evaluated using CellTiter-Glo® 2.0(Promega) following manufacturer protocols. Data was analyzed inCalcuSyn utilizing the median effect principle of presented byChou-Talalay and visualized using GraphPad Prism Software. Keyparameters assessed were combination index and dose reduction index.

We found the combination of Compound 101 with SOC chemotherapy(gemcitabine or carboplatin in SCLC, oxaliplatin in CRC, or capecitabinein HR+ breast cancer) showed synergy and was superior to either agentalone. The combination of Compound 101 with the targeted agenttrametinib, a selective MAPK pathway inhibitor approved for thetreatment of BRAF p.V600E mutant melanoma and NSCLC, show significantsynergy in BRAF p.V600E mutant CRC as well as in KRAS p.G12V mutant CRC,which harbors a different mutation within the MAPK pathway. Thecombination of Compound 101 with the targeted agent alpelisib, aselective PIK3CA inhibitor approved for the treatment of PIK3CA mutantHR+BC, showed significant synergy in both HR+ cell lines representingthe two most common activating mutation of PIK3CA (p.E545K andp.H1047R). All synergy was determined using CalcuSyn utilizing themedian effect principle of presented by Chou-Talalay and visualizedusing GraphPad Prism Software. Combination effect is reflected by shiftin IC50 of Compound 101 with addition of carboplatin or oxaliplatin orincreased antiproliferative effect with lower amounts of either singleagent. This is visualized in the isobolograms of FIG. 5, where pointsbelow the diagonal line reflect synergy.

Example 12. Deep and Sustained Responses to Compound 101 in TNBC, HGSOC,and SCLC PDX Models

We evaluated TGI in 12 different PDX models (Crown Biosciences) invarious tumor indications with PDXs representing SCLC (n=5; LU5180,LU5178, LU5192, LU5173, LU5210), TNBC (n=4; BR5010, BR1458, BR5399,BR10014) and HGSOC (n=3; OV15398, OV5392, OV15631). Dosing was initiatedwhen tumors were 150-300 mm³. Mice were treated with either Compound101, QD (6 or 10 mg/kg once daily, by mouth) or BID (3 or 5 mg/kg twicedaily, by mouth) over the course of 21 days, followed by 21 days ofobservation. TGI was calculated on the last day of dosing using theformula: TGI (V_(c1)−V_(t1))/(V_(c0)−V_(t0)), where V_(c1) and V_(t1)are the mean volumes of control and treated groups at the time of tumorextraction, while V_(c0) and V_(t0) are the same groups at the start ofdosing.

To perform whole exome sequencing (WES), we isolated DNA from passagematched tumors using DNeasy® Blood and Tissue Kit via manufacturerprotocol and sent it to Wuxi Aptec for WES using Agilent's SureSelectXTHuman All Exon V6 kit. Samples were sequenced to a depth of ˜300×. Readswere trimmed to remove adapter sequences via Skewer (v0.2.1). Reads werethen mapped and further processed using Sentieon tools: BWA, DeDup,Realigner, and QualCal (v201808.03). Variants were called usingSentieon's Haplotyper tool, and initial annotations were performed usingEnsembl's Variant Effect Predictor (VEP, release_96.2). FATHMM-MLK wasalso used to annotate variant effects. Variants that met the followingqualifications were included in sample characterizations: (1) variant islocated in a protein-coding gene; (2) variant affects protein sequenceor results in a frameshift; (3) missense mutations are classified asdamaging by SIFT, PolyPhen, or FATHMM-MLK (≥0.75); (4) variant allelefrequency is ≥10%. Copy-number (CN) variation across capture regionswere called using CNVkit (v0.9.1), and CNs for individual genes werecalculated by using the mean CN across its capture regions. For modelLU5210 mutation/CNV data was made available from WES data provided bythe PDX vendor (Crown Biosciences Inc.).

At these doses, Compound 101 induced at least 50% TGI at the end of the21-day dosing period in all models. In a subset of models (58%, 7/12),Compound 101 responses were deep (>95% TGI or regression) and sustained,with no evidence of tumor regrowth for 21 days after treatmentdiscontinuation (see FIG. 6). Compound 101 was well tolerated, with noevident body weight loss at all once-daily doses tested, indicating thatthe MTD is above 10 mg/kg once daily in tumor-bearing mice. Deep andsustained responses were observed in each indication tested and wereassociated with alterations in the RB pathway including RB1 deletion ormutation, CDKN2A deletion, or CCNE1 amplification (FIG. 7). Theseresults highlight the therapeutic potential of Compound 101 in TNBC,HGSOC, and SCLC, particularly in tumors with RB pathway alterations andaberrant cell cycle control.

It should it be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. Where rangesare given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, that there are many equivalents tothe specific embodiments of the disclosure described and claimed herein.Such equivalents are intended to be encompassed by the following claims.

1. A method of treating a selected patient, the method comprisingadministering a therapeutically effective amount of a compound ofstructural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the compound orthe pharmaceutically acceptable salt thereof is optionally within apharmaceutical composition; R¹ is methyl or ethyl; R² is methyl orethyl; R³ is 5-methylpiperidin-3-yl, 5,5-dimethylpiperidin-3-yl,6-methylpiperdin-3-yl, or 6,6-dimethylpiperidin-3-yl, wherein one ormore hydrogen atoms in R³ is optionally replaced by deuterium; R⁴ is—CF₃ or chloro; and the selected patient has been determined to have acancer in which (a) a gene selected from RB1, RBL1, RBL2, CDKN2A,CDKN2B, CDKN2C, CDKN2D, CDKN1A, CDKN1B, CDKN1C, and FBWX7 is mutated, isgenetically deleted, contains an epigenetic alteration, is translocated,is transcribed at a level equal to or below a pre-determined threshold,or encodes a protein that is translated at a level equal to or below apre-determined threshold or has decreased activity relative to areference standard; (b) a gene selected from E2F1, E2F2, E2F3, E2F4,E2F5, E2F6, E2F7, E2F8, CDK1, CDK2, CDK4, CDK6, CCNA1, CCNB1, CCND1,CCND2, CCND3, CCNE1, CCNE2, and BRAF is mutated, is genetically gainedor amplified, contains an epigenetic alteration, is translocated,transcribed at a level equal to or above a pre-determined threshold, orencodes a protein that is translated at a level equal to or above apre-determined threshold or has increased activity relative to areference standard; or (c) the gene Bcl2-like 1 is mutated, contains anepigenetic alteration, is translocated, is transcribed at a level equalto or below a pre-determined threshold, or encodes a BCL-xL protein thatis translated at a level equal to or below a pre-determined threshold orhas decreased activity relative to a reference standard.
 2. The methodof claim 1, wherein (i) R¹ is methyl and R² is methyl or (ii) R¹ ismethyl and R² is ethyl.
 3. The method of claim 1, wherein (i) R¹ isethyl and R² is ethyl or (ii) R⁴ is —CF₃.
 4. The method of claim 1,wherein R⁴ is chloro.
 5. The method of claim 1, wherein R³ is5-methylpiperidin-3-yl.
 6. The method of claim 1, wherein R³ is5,5-dimethylpiperidin-3-yl.
 7. The method of claim 1, wherein R³ is6-methylpiperdin-3-yl.
 8. The method of claim 1, wherein R³ is6,6-dimethylpiperidin-3-yl.
 9. The method of claim 1, wherein thecompound has structural Formula (Ia):

is a pharmaceutically acceptable salt thereof, wherein R³ is

10.-12. (canceled)
 13. The method of claim 9, wherein the compound is:

or is a pharmaceutically acceptable salt of any one of the foregoingcompounds.
 14. The method of claim 13, wherein the compound is

or a pharmaceutically acceptable salt thereof. 15.-18. (canceled) 19.The method of claim 1, wherein the cancer is a blood cancer, a breastcancer, Ewing's sarcoma, fallopian tube cancer, a GI tract cancer, aglioma, a lung cancer, melanoma, an osteosarcoma, an ovarian cancer, apancreatic cancer, a primary peritoneal cancer, prostate cancer,retinoblastoma, or a squamous cell cancer of the head or neck. 20.(canceled)
 21. The method of claim 19, wherein the patient hasundergone, is presently undergoing, or is prescribed treatment with aBcl-2 inhibitor.
 22. The method of claim 21, wherein the Bcl-2 inhibitoris venetoclax and/or wherein the patient has a breast cancer, a bloodcancer, an ovarian cancer, or a lung cancer.
 23. The method of claim 19,wherein the patient has been determined to have a cancer in which (a)RB1 or CDKN2A is mutated, contains an epigenetic alteration, istranslocated, is transcribed at a level equal to or below apre-determined threshold, or encodes a protein that is translated at alevel equal to or below a pre-determined threshold or has decreasedactivity relative to a reference standard; and/or (b) CDK6, CCND2, orCCNE1 is mutated, has a copy number alteration, contains an epigeneticalteration, is translocated, transcribed at a level equal to or above apre-determined threshold, or encodes a protein that is translated at alevel equal to or above a pre-determined threshold or has increasedactivity relative to a reference standard.
 24. The method of claim 19,wherein the patient has undergone, is presently undergoing, or isprescribed treatment with a selective estrogen receptor modulator(SERM), a selective estrogen receptor degrader (SERD), a PARP inhibitor,or a platinum-based therapeutic agent.
 25. The method of claim 24,wherein the patient has undergone, is presently undergoing, or isprescribed treatment with a SERM or SERD and has an HR+ breast cancer;the patient has undergone, is presently undergoing, or is prescribedtreatment with a PARP inhibitor and has breast cancer, fallopian tubecancer, a glioma, ovarian cancer, or primary peritoneal cancer; or thepatient has undergone, is presently undergoing, or is prescribedtreatment with a platinum-based therapeutic agent and has an ovariancancer.
 26. The method of claim 19, wherein the patient has undergone,is presently undergoing, or is prescribed treatment with a BET inhibitorwith a CDK4/6 inhibitor; with a FLT3 inhibitor; or with a MEK inhibitor.27. The method of claim 26, wherein the patient who has undergone, ispresently undergoing, or is prescribed treatment with the CDK4/6inhibitor has a breast cancer, a pancreatic cancer, or a squamous cellcancer of the head or neck; the patient who has undergone, is presentlyundergoing, or is prescribed treatment with the FLT3 inhibitor has ablood cancer; or the patient who has undergone, is presently undergoing,or is prescribed treatment with the BET inhibitor has a breast cancer, ablood cancer, Ewing's sarcoma, or an osteosarcoma.
 28. The method ofclaim 1, wherein the patient has undergone, is presently undergoing, oris prescribed treatment with a second anti-cancer agent.
 29. The methodof claim 28, wherein the second anti-cancer agent is a Bcl-2 inhibitor aCDK9 inhibitor; a hormone receptor degradation agent; a Flt3 (FMS-liketyrosine kinase 3) inhibitor; a PARP inhibitor; a BET inhibitor; aplatinum-based therapeutic agent; a CDK4/6 inhibitor; a MEK inhibitor;or a phosphoinositide 3-kinase (PI3 kinase) inhibitor.
 30. The method ofclaim 29, wherein the Bcl-2 inhibitor is venetoclax, the PARP inhibitoris olaparib or niraparib, the platinum-based anti-cancer agent iscarboplatin or oxaliplatin, the CDK4/6 inhibitor is palbociclib,ribociclib, abemaciclib, or trilaciclib, and the hormone receptordegradation agent is fulvestrant.